Nucleic acid melting analysis with saturation dyes

ABSTRACT

Methods are provided for nucleic acid analysis wherein a target nucleic acid is mixed with a dsDNA binding dye to form a mixture. Optionally, an unlabeled probe is included in the mixture. A melting curve is generated for the target nucleic acid by measuring fluorescence from the dsDNA binding dye as the mixture is heated. Dyes for use in nucleic acid analysis and methods for making dyes are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/462,904, filed Aug. 19, 2014, which is a continuation-in-part of U.S.patent application Ser. No. 11/931,174, filed Oct. 31, 2007, which is acontinuation of U.S. patent application Ser. No. 10/827,890 (now U.S.Pat. No. 7,387,887), filed Apr. 20, 2004. All of the aforementionedapplications are incorporated by reference herein in their entirety.

BACKGROUND

Field of the Invention

The present invention relates to double stranded nucleic acid bindingdyes and methods of performing nucleic acid analysis in the presence ofa double-stranded nucleic acid binding dye.

Background of the Invention

Methods for analyzing DNA sequence variation can be divided into twogeneral categories: 1) genotyping for known sequence variants and 2)scanning for unknown variants. There are many methods for genotypingknown sequence variants, and single step, homogeneous, closed tubemethods that use fluorescent probes are available (Lay M J, et al.,Clin. Chem 1997; 43:2262-7). In contrast, most scanning techniques forunknown variants require gel electrophoresis or column separation afterPCR. These include single-strand conformation polymorphism (Orita O, etal., Proc Natl Acad Sci USA 1989; 86:2766-70), heteroduplex migration(Nataraj A J, et al., Electrophoresis 1999; 20:1177-85), denaturinggradient gel electrophoresis (Abrams E S, et al., Genomics 1990;7:463-75), temperature gradient gel electrophoresis (Wartell R M, etal., J Chromatogr A 1998; 806:169-85), enzyme or chemical cleavagemethods (Taylor G R, et al., Genet Anal 1999; 14:181-6), as well as DNAsequencing. Identifying new mutations by sequencing also requiresmultiple steps after PCR, namely cycle sequencing and gelelectrophoresis. Denaturing high-performance liquid chromatography (XiaoW, et al., Hum Mutat 2001; 17:439-74) involves injecting the PCR productinto a column.

Recently, homogeneous fluorescent methods have been reported formutation scanning. SYBR® Green I (Molecular Probes, Eugene, Oreg.) is adouble strand-specific DNA dye often used to monitor product formation(Wittwer C T, et al., BioTechniques 1997; 22:130-8) and meltingtemperature (Ririe K M, et al., Anal. Biochem 1997; 245:154-60) inreal-time PCR. The presence of heterozygous single base changes havebeen detected in products up to 167 bp by melting curve analysis withSYBR® Green I (Lipsky R H, et al., Clin Chem 2001; 47:635-44). However,subsequent to amplification and prior to melting analysis, the PCRproduct was purified and high concentrations of SYBR® Green I wereadded. The concentration of SYBR® Green I used for detection in thismethod inhibits PCR (Wittwer C T, et al., BioTechniques 1997; 22:130-1,134-8); thus, the dye was added after amplification. A dye that could beused to detect the presence of genetic variation including heterozygoussingle base changes and could be added prior to PCR would be desirable.

Single nucleotide polymorphisms (SNPs) are by far the most commongenetic variations observed in man and other species. In thesepolymorphisms, only a single base varies between individuals. Thealteration may cause an amino acid change in a protein, alter rates oftranscription, affect mRNA spicing, or have no apparent effect oncellular processes. Sometimes when the change is silent (e.g., when theamino acid it codes for does not change), SNP genotyping may still bevaluable if the alteration is linked to (associated with) a uniquephenotype caused by another genetic alteration.

There are many methods for genotyping SNPs. Most use PCR or otheramplification techniques to amplify the template of interest.Contemporaneous or subsequent analytical techniques may be employed,including gel electrophoresis, mass spectrometry, and fluorescence.Fluorescence techniques that are homogeneous and do not require theaddition of reagents after commencement of amplification or physicalsampling of the reactions for analysis are attractive. Exemplaryhomogeneous techniques use oligonucleotide primers to locate the regionof interest and fluorescent labels or dyes for signal generation.Illustrative PCR-based methods are completely closed-tubed, using athermostable enzyme that is stable to DNA denaturation temperature, sothat after heating begins, no additions are necessary.

Several closed-tube, homogeneous, fluorescent PCR methods are availableto genotype SNPs. These include systems that use FRET oligonucleotideprobes with two interacting chromophores (adjacent hybridization probes,TaqMan probes, Molecular Beacons, Scorpions), single oligonucleotideprobes with only one fluorophore (G-quenching probes, Crockett, A. O.and C. T. Wittwer, Anal. Biochem. 2001; 290:89-97 and SimpleProbes,Idaho Technology), and techniques that use a dsDNA dye instead ofcovalent, fluorescently-labeled oligonucleotide probes. The dyetechniques are attractive because labeled oligonucleotide probes are notrequired, allowing for reduced design time and cost of the assays.

Two techniques for SNP typing using dsDNA dyes have been published.Allele-specific amplification in the presence of dsDNA dyes can be usedto genotype with real-time PCR (Germer S, et al., Genome Research 2000;10:258-266). In the method of the Germer reference, two allele-specificprimers differ at their 3′-base and differentially amplify one or theother allele in the presence of a common reverse primer. While nofluorescently-labeled oligonucleotides are needed, genotyping requiresthree primers and two wells for each SNP genotype. In addition, areal-time PCR instrument that monitors fluorescence each cycle isnecessary.

The other dye-based method does not require real-time monitoring, needsonly one well per SNP genotype, and uses melting analysis (Germer, S,et. al., Genome Research 1999; 9:72-79). In this method, allele-specificamplification is also used, requiring three primers, as with theprevious Germer method. In addition, one of the primers includes aGC-clamp tail to raise the melting temperature of one amplicon, allowingdifferentiation by melting temperature in one well. Fluorescence ismonitored after PCR amplification, and real-time acquisition is notrequired.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a method is provided thatrequires only a standard PCR mixture, including reagents, primers, andthe simple addition prior to PCR of a “saturating” double-stranded (ds)DNA binding dye or a novel dsDNA binding dye according to thisdisclosure. For purposes of this disclosure, a “saturating” dye is a dyethat does not significantly inhibit PCR when present at concentrationsthat provide maximum fluorescence signal for an amount of dsDNAtypically generated by PCR in the absence of dye, illustratively about10 ng/μL. Although the dyes are identified by their compatibility withPCR at near saturating concentrations, it is understood that the dyescan be used at much lower concentrations. During or subsequent toamplification, the dyes may be used to distinguish heteroduplexes andhomoduplexes by melting curve analysis in a similar fashion to whenlabeled primers are used. The identification of heteroduplexes andhomoduplexes may be used for a variety of analyses, including mutationscanning and genotyping. The term “scanning” refers to the process inwhich a nucleic acid fragment is compared to a reference nucleic acidfragment to detect the presence of any difference in sequence. Apositive answer indicating the presence of a sequence difference may notnecessarily reflect the exact nature of the sequence variance or itsposition on the nucleic acid fragment. The term “genotyping” includesthe detection and determination of known nucleic acid sequencevariances, including but not limited to, SNPs, base deletions, baseinsertions, sequence duplications, rearrangements, inversions, basemethylations, the number of short tandem repeats; and in the case of adiploid genome, whether the genome is a homozygote or a heterozygote ofthe sequence variance, as well as the cis/trans positional relationshipof two or more sequence variances on a DNA strand (haplotyping).Optionally, one or more unlabeled probes may be added to the mixture atany time prior to melting curve analysis.

The term “unlabeled probe” refers to an oligonucleotide orpolynucleotide that is not covalently linked to a dye and that isconfigured to hybridize perfectly or partially to a target sequence. Thedye that is present in the mixture is free to bind to or disassociatefrom the unlabeled probe, particularly as the probe hybridizes to andmelts from the target sequence. The terms “oligonucleotide” and“polynucleotide” as used herein include oligomers and polymers ofnatural or modified monomers or linkages including deoxyribonucleosides,ribonucleosides, protein nucleic acid nucleosides, and the like that arecapable of specifically binding to a target polynucleotide bybase-pairing interactions. Optionally, the unlabeled probe may bemodified with one or more non-fluorescent moieties, such as but notlimited to non-fluorescent minor-groove binders, biotin, spacers,linkers, phosphates, base analogs, non-natural bases, and the like.

In another aspect of this invention, various dsDNA binding dyes areidentified. The dsDNA binding dyes of the present invention are capableof existing at sufficiently saturating conditions with respect to theDNA during or after amplification, while minimizing the inhibition ofPCR. For example, at maximum PCR-compatible concentrations, the dsDNAbinding dye has a percent saturation of at least 50%. In otherembodiments, the percent saturation is at least 80%, and moreparticularly, at least 90%. In yet other embodiments, the percentsaturation is at least 99%. It is understood that the percent saturationis the percent fluorescence compared to fluorescence of the same dye atsaturating concentrations, i.e. the concentration that provides thehighest fluorescence intensity possible in the presence of apredetermined amount of dsDNA. Illustratively, the predetermined amountof dsDNA is 100 ng/10 μL which is the amount of DNA produced at the endof a typical PCR at plateau. It is further understood that dyepreparations may contain impurities that inhibit amplification. Suchimpurities should be removed prior to a determination of the percentsaturation. It is also understood that the measurement of fluorescenceintensity for percent saturation is performed at the wavelength that iswell matched for the detection of dye bound to dsDNA, and if possible,not at wavelengths that will detect high background fluorescence fromfree dye or secondary forms of dye binding which may occur at a highdye-to-bp ratio (e.g. binding of dye to the dsDNA-dye complex or tosingle-stranded nucleic acids).

In yet another aspect of the present invention, the dsDNA binding dyehas greater than 50% saturation at maximum PCR-compatibleconcentrations, and has excitation/emission spectra that would notsuggest compatibility with standard real-time PCR instruments.“Standard” instruments for real-time PCR analysis have an excitationrange of about 450-490 nm and an emission detection range of about510-530 nm. It has been found that certain “blue” dyes are compatiblewith these systems, although their excitation/emission spectra wouldsuggest otherwise. Thus, in this aspect of the invention a method isprovided for analysis during or subsequent to PCR using a standardreal-time PCR instrument and a dsDNA binding dye having an excitationmaximum in the range of 410-465 nm, more particularly in the range of430-460 nm, and having an emission maximum in the range of 450-500 nm,more particularly in the range of 455-485 nm, as measured in PCR bufferin the presence of dsDNA. Suitable instrumentation may use theexcitation/detection ranges above, or may be modified according to theexcitation/emission maxima of the dyes. Suitable ranges for detection ofthe “blue” dyes of this invention as well as for detection oftraditional dyes such as fluorescein and SYBR® Green I may include440-470 nm for excitation and 500-560 for detection. It is noted thatwhile many of these dyes are suitable for use with standard real-timePCR instruments and melting instrumentation, adjustment of the optics tobetter match the excitation/emission spectra of these dyes may furtherimprove their sensitivity for use in quantitative or qualitativeamplification analysis.

In yet another aspect of this invention, scanning or genotyping isperformed by melting curve analysis in the presence of one or moreunlabeled probes and a double-stranded binding dye. The melting curveanalysis may take place during or subsequent to amplification, or in theabsence of amplification. The dye may be a saturating dye or a novel dyeaccording to this disclosure.

While the examples provided herein are directed to melting curveanalysis, it is understood that the dyes of the present invention can beused for a variety of real-time quantitative PCR analyses, includingquantification of the nucleic acid, determination of initialconcentration, testing for the presence of a nucleic acid, multiplexingwith labeled probes, and other PCR-based methods.

Furthermore, while reference is made to PCR, other methods ofamplification may be compatible with the dyes of this invention. Suchsuitable procedures include strand displacement amplification (SDA);nucleic acid sequence-based amplification (NASBA); cascade rollingcircle amplification (CRCA), Q beta replicase mediated amplification;isothermal and chimeric primer-initiated amplification of nucleic acids(ICAN); transcription-mediated amplification (TMA), and the like.Asymmetric PCR may also be used. Therefore, when the term PCR is used,it should be understood to include variations on PCR and otheralternative amplification methods. Amplification methods that favoramplification of one strand over the other are particularly well suitedfor melting curve analysis using unlabeled probes.

Moreover, while reference is made to amplification, it is understoodthat the melting curve analysis of the present invention may beperformed on nucleic acid samples that have been obtained withoutamplification.

Additionally, it is understood that the dsDNA binding dyes includeintercalators, as well as other dyes that bind to nucleic acids, as longas the dye differentially binds to double-stranded and single-strandednucleic acids, or otherwise produces a differential signal based on thequantity of double-stranded nucleic acid.

Thus, in one embodiment of this invention, novel dyes are presented. Thenovel dyes, which may or may not be saturating dyes, may be used duringor subsequent to amplification, or may be used during melting curveanalysis in the presence or absence of amplification. Illustratively,the novel dyes have the formula:

wherein

the moiety

represents an optionally-substituted fused monocyclic or polycyclicaromatic ring or an optionally-substituted fused monocyclic orpolycyclic nitrogen-containing heteroaromatic ring;

X is oxygen, sulfur, selenium, tellurium or a moiety selected fromC(CH₃)₂ and NR¹, where R¹ is hydrogen or C₁₋₆ alkyl;

R² is selected from the group consisting of C₁₋₆ alkyl, C₃₋₈ cycloalkyl,aryl, aryl(C₁₋₂ alkyl), hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono anddialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate,alkylenecarboxamide, alkylenesulfonate, optionally substituted cyclicheteroatom-containing moieties, and optionally substituted acyclicheteroatom-containing moieties;

t=0 or 1;

Z is a charge selected from 0 or 1;

R³, R⁹, and R¹⁰ are each independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, and arylcarbonyl;

n=0, 1, or 2; and

Q is an heterocycle selected from the group of structures consisting of:

wherein R⁴, R⁵, R⁶, R⁷, and R⁸ are independently selected from the groupconsisting of hydrogen, halogen, alkyl, cycloalkyl, heteroalkyl,heterocycloalkyl, alkenyl, polyalkenyl, alkynyl, polyalkynyl,alkenylalkynyl, aryl, heteroaryl, alkoxy, alkylthio, arylcarbonylthio,cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio,dialkylamino, cycloalkylthio, cycloheteroalkylthio,trialkylammoniumalkylthio, and nucleosidylthio, each of which may beoptionally substituted; an acyclic heteroatom-containing moiety, acyclic heteroatom-containing moiety, a BRIDGE-DYE, and a reactive group,each of which optionally includes a quaternary ammonium moiety.

Illustratively, in one embodiment at least one of R⁴, R⁵, R⁶, R⁷, and R⁸is selected from the group consisting of arylcarbonylthio,cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio,cycloalkylthio, cycloheteroalkylthio, trialkylammoniumalkylthio, andnucleosidylthio, each of which may be optionally substituted. In anotherembodiment, the dye is selected from the group consisting of N7, O7, P7,Q7, R7, S7, T7, U7, V7, W7, X7, Z7, K8, P8, T8, W8, X8, Z8, A9, C9, G9,19, J9, K9, L9, M9, N9, O9, P9, Q9, R9, and A10. In one method, thesedyes are used in PCR amplification. In another method, these dyes areused with a target nucleic acid and an unlabeled probe in melting curveanalysis. These dyes may be used with various other methods describedherein.

In a further embodiment of this invention, a method is provided fornucleic acid analysis comprising the steps of mixing a target nucleicacid with a saturating dsDNA binding dye and at least one unlabeledprobe configured to hybridize to a portion of the target nucleic acid,to form a mixture, allowing the unlabeled probe to hybridize to thetarget nucleic acid to form a probe/target duplex, generating a meltingcurve for the probe/target duplex by measuring fluorescence from thedsDNA binding dye as the mixture is heated, and analyzing the shape ofthe melting curve. Illustratively, the shape of the melting curve may beanalyzed by generating a derivative melting curve, illustratively byanalyzing the shape and location of one or more melting peaks on thederivative melting curve. The analysis optionally may take place duringor subsequent to amplification of the target nucleic acid. Thesaturating dyes described above or other saturating dyes may be used forthis method.

In yet another embodiment of this invention, a kit is provided foranalyzing a target nucleic acid, the kit comprising an unlabeled probeconfigured to hybridize at least partially to the target nucleic acid,and a saturating dsDNA binding dye. Optionally, the kit may includeother components, illustratively a thermostable polymerase andoligonucleotide primers configured for amplifying the target nucleicacid.

In a further embodiment of this invention, a method of detectingmutations in the c-kit gene is provided comprising providing anamplification mixture comprising a nucleic acid sample, one or morepairs of primers configured for amplifying a locus of the c-kit gene, athermostable polymerase, and a saturating dsDNA binding dye, amplifyingthe nucleic acid sample to generate an amplicon, melting the amplicon togenerate a melting curve, and analyzing the shape of the melting curve.Illustratively, the primers include any or all of the primers selectedfrom the group consisting of GATGCTCTGCTTCTGTACTG (SEQ ID NO. 40) andGCCTAAACATCCCCTTAAATTGG (SEQ ID NO. 41); CTCTCCAGAGTGCTCTAATGAC (SEQ IDNO. 42) and AGCCCCTGTTTCATACTGACC (SEQ ID NO. 43); CGGCCATGACTGTCGCTGTAA(SEQ ID NO. 44) and CTCCAATGGTGCAGGCTCCAA (SEQ ID NO. 45); andTCTCCTCCAACCTAATAGTG (SEQ ID NO. 46) and GGACTGTCAAGCAGAGAAT (SEQ ID NO.47).

In still another embodiment, a method for nucleic acid analysis isprovided, comprising the steps of mixing a target nucleic acid with asaturating dsDNA binding dye to form a mixture, generating a meltingcurve for the target nucleic acid by measuring fluorescence from thedsDNA binding dye as the mixture is heated, including in the mixture asecond nucleic acid configured to hybridize with a portion of the targetnucleic acid, the second nucleic acid being smaller than the targetnucleic acid and having a melting temperature different from the targetnucleic acid, and allowing the second nucleic acid to hybridize to theportion of the target nucleic acid, melting the second nucleic acid fromthe first nucleic acid, and analyzing the shape of the melting curve. Inone embodiment, the second nucleic acid is an unlabeled probe that maybe added prior to or subsequent to generating the melting curve for thetarget nucleic acid, whereas in another embodiment, the secondnucleotide is a smaller amplicon illustratively that may be produced ina single mixture with amplification of the target nucleic acid.

An additional embodiment of the invention is a method of PCR analysiscomprising the steps of mixing a dsDNA binding dye with a samplecomprising an unknown initial quantity of a target nucleic acid andprimers configured for amplifying the target nucleic acid, to form amixture, amplifying the target nucleic acid in the presence of the dsDNAbinding dye, monitoring fluorescence of the dsDNA binding dye throughouta temperature range during a plurality of amplification cycles togenerate a plurality of melting curves, and using the melting curves toquantify the initial quantity of the target nucleic acid. Unlabeledprobes and/or saturating dyes may be used during amplification.

Additional features of the present invention will become apparent tothose skilled in the art upon consideration of the following detaileddescription of illustrative embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows genotyping of the Factor V Leiden using dye S5. Thenegative first derivative (−dF/dT) of the melting curve is shown.

FIG. 2 shows the effect of cooling rates prior to melting analysis onthe detection of heteroduplexes.

FIG. 3 shows the effect of heating rates during melting analysis on thedetection of heteroduplexes.

FIGS. 4A-B are normalized, high resolution melting curves of allpossible SNP genotypes at one position using an engineered plasmid.Three samples of each genotype were analyzed and included fourhomozygotes (FIG. 4A, ———— A/A,

, T/T,

C/C,

G/G) and six heteroduplexes (FIG. 4B,

A/T,

A/C,

C/T,

A/G, ———— G/T, ——— - - ——— C/G).

FIGS. 5A-D show a comparison of genotyping methods; FIG. 5A shows thecystic fibrosis map in which the position of an optional label on aprimer is marked (star), FIG. 5B shows genotyping using a labeledprimer, FIG. 5C shows genotyping using dye S5, and FIG. 5D shows anattempt to genotype using SYBR® Green I (Homozygotes: ——— - - ——— wt,———— F508del; Heterozygotes: —— • —— F508del, ——— - ——— I507del, - - - -F508C).

FIG. 6 shows genotyping using dye S5 on longer amplicons (——— - - ———homozygote (TT), ———— homozygote (CC), ——— • ——— heterozygote (TC)). Themelting curves for three individuals (not the derivatives) are shown.

FIGS. 7A-B shows derivative melting curves of DNA mixtures using SYBR®Green I (FIG. 7A) and dye S5 (FIG. 7B).

FIG. 8 demonstrates the non-linearity of fluorescence change whenmultiple DNA species are present. Dye S5 (open circles) and SYBR® GreenI (closed squares) are shown.

FIGS. 9A-B show dye titrations to determine saturation percentages, inFIG. 9A, ♦-SYBR® Green, ▪-SYBR® Gold, ▴-Pico Green, in FIG. 9B, ∘-dyeS5, ▪-SYTOX® Green. Illustrative PCR ranges for SYBR® Green I and dye S5are indicated by the shaded box.

FIG. 10 illustrates the effect of dye concentrations on meltingtemperature.

FIGS. 11A-B show the excitation and emission spectra for dye S5 (FIG.11A) and SYBR® Green I (FIG. 11B).

FIGS. 12A-D show high resolution melting curve analysis of quadruplicatesamples of six different genotypes within a 110 bp fragment ofbeta-globin (——— - ——— SS, ———— AA, ——— - - ——— CC,

SC,

AC,

AS); FIG. 12A shows raw data obtained from high resolution melting ofquadruplicate samples of each genotype; FIG. 12B shows normalized highresolution melting curves of the quadruplicate samples of the sixgenotypes; FIG. 12C shows temperature-shifted, normalized, highresolution melting curves of the quadruplicate samples of the sixgenotypes. The samples were temperature shifted to overlay the curvesbetween 5 and 10% fluorescence; FIG. 12D shows fluorescence differencecurves obtained from the data of FIG. 12C. Each difference curve wasobtained by subtracting each sample from the normal (AA) curve to obtainthe difference data. While quadruplicate samples were run, due tooverlap, fewer than four samples appear in some instances.

FIG. 13A shows melting curve analysis of duplicate samples of threegenotypes of a 544 bp fragment of the human 5-Hydroxytryptamine receptor2A (HTR2A) gene (——— - ——— TC, ———— CC, ——— - - ———TT). The data havebeen normalized and temperature shifted using the portion between 10 and20% fluorescence. A theoretical melting map of the homoduplex is shownas FIG. 13B. The position of the single nucleotide polymorphism ismarked (X).

FIG. 14 shows a difference curve of six genotypes of a 612 bp fragmentof the cystic fibrosis transmembrane conductance regulator (CFTR) gene.The plots have been normalized, temperature shifted by superimposing theportion between 30 and 40% fluorescence, and subtracted from one of thewild type plots.

FIG. 15 shows the pedigree of CEPH referenced Utah family 1331. Genotypeof HLA-A of Utah family 1331 are as follows: A:02011; B:3101; C:2402101;D:03011; E:01011. Each individual is numbered. Female (circle); male(square).

FIGS. 16A and B show the melting curve of Utah family 1331 members. Sixdifferent melting curves representing six genotypes in HLA-A exon 2exist among 17 family members. FIG. 16A shows the full melting curve andFIG. 16B shows an enlarged portion (shown in square in 16A) with thedesignation of genotype, and designation of individuals in parentheses.

FIG. 17 shows the determination of genotypes of two samples by mixing(———— BM15, ——— - - ——— BM16, - - - - BM15+BM16). Two homozygous samplesBM15(0101) and BM16(0201) have a 15-bp difference on the HLA-A exon 2.The melting curve of BM15 and BM16 are similar when taken separately,but when mixed, the 15-bp mismatch shifts the melting curve.

FIGS. 18A and B show the results of an optimization experiment forgenotyping with an unlabeled probe following asymmetric PCR. FIG. 18Ashows the results of amplification with various ratios of primers (

symmetric (0.5 μM of each primer); • • • • • symmetric (no templatecontrol); ———— (light) 0.05 μM sense primer and 0.5 μM reverse primer;———— (heavy) 0.5 μM sense primer and 0.05 μM reverse primer; - - - -(light) 0.5 μM sense primer and 0.025 μM reverse primer; - - - - (heavy)0.025 μM sense primer and 0.5 μM reverse primer; —— - —— (light) 0.5 μMsense primer and 0.01 μM reverse primer; —— - —— (heavy) 0.01 μM senseprimer and 0.5 μM reverse primer). FIG. 18B is a derivative meltingcurve showing melting peaks (

symmetric (0.5 μM of each primer); - - - - 0.05 μM sense primer and 0.5μM reverse primer; ———— 0.5 μM sense primer and 0.05 μM reverse primer).

FIG. 19 is similar to FIG. 18B, showing melting peaks after asymmetricamplification (

symmetric (0.5 μM of each primer); solid line 0.5 μM sense primer and0.05 μM reverse primer; - - - - 0.5 μM sense primer and 0.025 μM reverseprimer; ———— - ———— - 0.5 μM sense primer and 0.01 μM reverse primer).

FIG. 20 is a derivative melting curve showing melting peaks forunlabeled probes ranging in length from 14 to 30 nucleotides.

FIGS. 21A-D are derivative melting curves showing melting peaks in atest system for unlabeled probes. FIG. 21A shows derivative meltingcurves for each of the four homozygotes using dye D6 FIG. 21B showsderivative melting curves for the A homozygote and the A/G, A/T, and A/Cheterozygotes using dye D6; FIG. 21C shows derivative melting curves forthe A homozygote and the A/G, A/T, and A/C heterozygotes using SYBR®Green I; and FIG. 21D shows derivative melting curves for the Ahomozygote and the G/T, C/G, and C/T heterozygotes using dye D6.

FIGS. 22A-B are derivative melting curves showing melting peaks forvarious cystic fibrosis mutations using an unlabeled probe.

FIG. 23 is a derivative melting curve showing melting peaks for a cysticfibrosis SNP mutation using two different unlabeled probes.

FIG. 24 is a derivative melting curve showing melting peaks for cysticfibrosis mutations F508del and Q493V using two unlabeled probes in thesame reaction.

FIGS. 25 A-B are melting curves for a PCR amplicon that includes thecystic fibrosis G542X locus, in which the samples were simultaneouslyscanned for the mutation by amplicon melting and genotyped by probemelting. FIG. 25A shows a fluorescence versus temperature plot for databetween 75° C. and 83° C. (the amplicon melting profile). The insetshows a magnified view of the portion of the curve indicated by thesquare. FIG. 25B shows a derivative plot of data between 58° C. and 72°C. (the probe melting profile), ——— wild type; • • • G542Xhomozygote; - - - G542X heterozygote.

FIG. 26 illustrates PCR parameters programmed on the LightCycler formonitoring unlabeled probe/target duplex melting during eachamplification cycle. Two PCR cycles are shown. Fluorescence wasmonitored continuously between annealing and extension (indicated by thesolid line) for each cycle.

FIG. 27 shows derivative melting curves obtained during each cycle ofPCR using an unlabeled probe and dye N7. The peak height increases withcycle number. The initial concentration of template DNA in this samplewas 10⁵ copies/10 μl.

FIGS. 28 A-D show analyses of fluorescence data obtained during eachcycle of PCR. FIG. 28A shows the cycle number versus fluorescence plotof data acquired at 61° C. (reflecting the amount of total dsDNA in thereaction, ▪) and at 73° C. (reflecting the amount of amplicon, □). FIG.28B shows the cycle number plotted against melting peak area (▴) andagainst the difference between the top of the melting peak and justbefore the melting transition calculated from the derivative data (Δ).FIG. 28C shows the cycle number versus melting peak area plot for threedifferent initial template concentrations (▴-10⁴ copies/10 μl; ▪-10⁵copies/10 μl; □-10⁶ copies/10 μl). FIG. 28D shows the log of the initialtemplate concentration plotted against the crossing point of each samplethat was derived from FIG. 28C.

FIGS. 29-32 show melting curves for genotyping various gastrointestinalstromal tumor (GIST) mutations, each comparing to normal wild typeamplicons.

FIG. 29 shows a heterozygous SNP (———— normal, - - - - GIST 1),

FIG. 30 shows a homozygous 12 bp deletion/SNP (———— normal, - - - - GIST2),

FIG. 31 shows a heterozygous tandem duplication (36 bp) (————normal, - - - - GIST 3), and

FIG. 32 shows a heterozygous deletion (54 bp) (———— normal, - - - - GIST4).

DETAILED DESCRIPTION

SYBR® Green I is a dye extensively used for melting analysis as it showsa large change in fluorescence during PCR (Wittwer C T, et al.,BioTechniques 1997; 22:130-1, 134-8; Wittwer C T, et al., Real-Time PCR.In: Persing D, et al., eds. Diagnostic Molecular Microbiology:Principles and Applications. ASM Press, 2004: in press). SYBR® Green Iwas first used in melting analysis to distinguish different PCR productsthat differed in Tm by 2° C. or more (Ririe K M, et al., Anal Biochem1997; 245:154-160). Subsequently, SYBR® Green I was used to identifydeletions (Aoshima T, et al., Clin Chem 2000; 46:119-22), genotypedinucleotide repeats (Marziliano N, et al., Clin Chem 2000; 46:423-5),and identify various sequence alterations (Lipsky R H, et al., Clin Chem2001; 47:635-44; Pirulli D, et al., Clin Chem 2000; 46:1842-4;Tanriverdi S, et al., J Clin Microbiol. 2002; 40:3237-44; Hladnik U, etal., Clin Exp Med. 2002; 2:105-8). However, the Tm difference betweengenotypes can be small and may challenge the resolution of currentinstruments. Indeed, it has been suggested that SYBR® Green I, “shouldnot be used for routine genotyping applications” (von Ahsen N, et al.,Clin Chem 2001; 47:1331-1332). Melting curve genotyping with commonlyused double-strand-specific DNA dyes can result an increased Tm withbroadening of the melting transition (Douthart R J, et al., Biochemistry1973; 12:214-20), and compression of the Tm difference between genotypes(FIG. 5D). These factors lower the potential of SYBR® Green I forgenotype discrimination.

Heterozygous DNA is made up of four different single strands that cancreate two homoduplex and two heteroduplex products when denatured andcooled. Theoretically, all four products have different Tms and themelting curve should be a composite of all four double-stranded tosingle-stranded transitions. However, double-strand-specific DNA dyesmay redistribute during melting (Aktipis S, et al., Biochemistry 1975;14:326-31), causing release of the dye from low melting heteroduplexesand redistribution to higher melting homoduplexes. Because SYBR® Green Iis not saturating at concentrations compatible with PCR (Wittwer C T, etal., BioTechniques 1997; 22:130-1, 134-8; FIG. 9), such redistributionis plausible and consistent with the absence of a heteroduplextransition (FIG. 5D).

The dyes of the present invention can be used for genotyping andscanning applications. When only one PCR product is amplified and thesequence is homozygous, only homoduplexes are formed. With the dyes ofthe present invention, Tm differences between different homoduplexgenotypes are not compressed (FIG. 5C), and clear differentiationbetween genotypes is possible, even for SNPs. The dyes of the presentinvention can also identify and distinguish multiple products present ina reaction, illustratively homoduplexes generated from amplification ofmultiple loci or multiple targets that are homozygous. In contrast, mostof the time only a few products can be observed with SYBR® Green I,presumably due to dye redistribution (see FIG. 7A).

When one or more heterozygous targets are amplified, heteroduplexproducts are readily observable with the dyes of the present invention.The ability to detect and identify heteroduplexes is particularly usefulfor detecting heterozygous genotypes as well as for scanning unknownmutations. This is not possible with conventional dsDNA dyes used inreal-time PCR, such as SYBR® Green I, SYBR® Gold, and ethidium bromide,where heteroduplex products are not observable.

Heteroduplex strands may re-associate with their perfect complement andform homoduplexes during melting. Because the concentration of productsat the end of PCR is high, this re-association happens rapidly.Re-association can be minimized by limiting the time the products arenear their melting temperatures, particularly between the Tms of theheteroduplex and homoduplex products. In addition to strandre-association during melting, the selective hybridization of a strandto either its perfect match, or to its mismatched complementary strand,is influenced by cooling rates. Under conditions presented herein,heteroduplex formation is most favored by rapid cooling and oftendisappears at rates slower than −0.1° C./s (FIG. 2). This is in contrastto denaturing HPLC techniques, where cooling rates are much slower(−0.01 to about −0.02° C./s), yet heteroduplexes are efficiently formed(Xiao W, et al., Hum Mutat 2001; 17:439-74). Perhaps the relative ratesof homoduplex and heteroduplex formation are strongly dependent onproduct size, and the results obtained using small amplicons may not betypical for the larger products more commonly used in dHPLC.

The discrimination between homozygous genotypes can be improved bymelting at slower rates, at the expense of greater analysis time. Onesource of potential error in melting curve genotyping is the effect ofDNA concentration on Tm. Using a random 100 bp amplicon of 50% GCcontent under PCR conditions, the difference in Tm between products at0.05 μM and 0.5 μM is about 0.7° C. (von Ahsen N, et al., Clin Chem2001; 47:1956-61; Wetmur J G, Crit Rev Biochem Mol Biol 1991;26:227-59). This change can be important when the Tms of differenthomozygous genotypes are very close. However, different PCR samples tendto plateau at the same product concentration, so post-amplificationconcentration differences are usually minimal. Also, it may be possibleto estimate amplicon concentrations by real-time fluorescence and adjustthe Tms for even greater genotyping precision. Alternatively, asymmetricPCR may be used to limit automatically the final concentration of PCRproduct.

With the dyes of the present disclosure, it is possible to distinguishall single base heterozygotes from homozygotes. In the detection ofheterozygotes, the absolute melting temperature and the influence of DNAconcentration are not as important as with methods involvingdifferentiation between homozygous genotypes. Heteroduplexes affect theshape of the melting curve, particularly at the “early,” low temperatureportion of the transition. Different melting curves can be temperaturematched by translating the X-axis to superimpose the “late,” hightemperature portion of the transition. The presence or absence ofheteroduplexes can then be inferred with greater accuracy. Thus, even insamples obtained without PCR amplification, attention to DNAconcentration may not be crucial.

Whatever the precision of the instrument, some genotypes will be nearlyidentical in Tm. One way to detect homozygous variants with the same Tmis to mix the variants together. The resulting heteroduplexes will meltat lower temperatures than the homoduplexes, displayed as a drop in thenormalized melting curves before the major melting transition.

Thus, using presently available PCR amplification devices, the dyes ofthe present invention can identify heteroduplexes in melting curvetransitions that cannot currently be identified using SYBR® Green I. Onepossible reason why SYBR® Green I cannot easily identify low meltingtransitions is shown in FIG. 7A. When several DNA fragments ofincreasing stability are present, the low temperature peaks are verysmall with SYBR® Green I compared to dyes such as dye S5 (structureshown in Example 1). During melting, SYBR® Green I may be released fromlow temperature duplexes, only to attach to duplexes that melt at highertemperatures. This causes each successive peak to be higher than thelast, with the lowest temperature peaks being very small, if observableat all. As seen in FIG. 7B, low temperature melting products are easilydetected with dye S5, but not by SYBR® Green I.

The advantages of using dye S5 have led to identification of other dsDNAdyes that are compatible with PCR and are suited for genotyping atPCR-compatible concentrations. Many of the dyes useful in the methods ofthe present invention belong to a family of cyanines. Cyanine dyes arethose dyes containing one or more divalent moieties “—C(R)═” arranged ina chain that link two nitrogen containing heterocycles. The group “R”may be hydrogen or any carbon substituent, and is illustrativelyhydrogen or alkyl, including C₁₋₆ alkyl, which may be optionallysubstituted. It is understood that in cyanine dyes where there is morethan one divalent moiety “—C(R)═” each “R” may be selectedindependently. Such cyanine dyes may be monomers or dimers, as furtherdefined by the illustrative general formulae herein described. Manycyanine variants, illustratively dyes in which the divalent moiety is═N—, —C(R)═N—, or the like, are also well suited. In addition to cyaninedyes, it is contemplated herein that other families of dsDNA bindingdyes are also useful in the PCR reaction mixtures, methods, andcompositions described herein, including but not limited tophenanthridinium intercalators and phenanthroline-basedmetallointercalators.

Illustrative dyes useful in the present PCR reaction and melting curvemixtures, methods, and compositions include, PO-PRO™-1, BO-PRO™-1, SYTO®43, SYTO® 44, SYTO® 45, SYTOX® Blue, POPO™-1, POPO™-3, BOBO™-1, BOBO™-3,LO-PRO™-1, JO-PRO™-1, YO-PRO®-1, TO-PRO®-1, SYTO® 11, SYTO® 13, SYTO®15, SYTO® 16, SYTO®20, SYTO® 23, TOTO™-3, YOYO®-3 (Molecular Probes,Inc., Eugene, Oreg.), GelStar® (Cambrex Bio Science Rockland Inc.,Rockland, Me.), thiazole orange (Aldrich Chemical Co., Milwaukee, Wis.)and various novel dyes described herein.

Illustrative cyanine dyes for use in the PCR reaction mixtures, methods,and compositions described herein also include monomers or dimers ofunsymmetrical cyanines having pyridinium, pyrimidinium, quinolinium,isoquinolinium, or purinium core structures, and those generallydescribed by Formula I:

wherein

the moiety

represents an optionally-substituted fused mono or polycyclic aromaticor nitrogen-containing heteroaromatic ring;

X is oxygen, sulfur, selenium, tellurium, or a group selected fromC(CH₃)₂ and NR¹, where R¹ is hydrogen or alkyl, including C₁₋₆ alkyl andC₂₋₆ alkyl;

R² is alkyl, including C₁₋₆ alkyl and C₂₋₆ alkyl, cycloalkyl, includingC₃₋₈ cycloalkyl, aryl, arylalkyl, including aryl(C₁₋₃ alkyl),hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono and dialkylaminoalkyl,trialkylammoniumalkyl, alkyl and arylcarbonyl, alkyl andarylcarboxamide, alkyl and arylsulfonyl, alkylenecarboxylate,alkylenecarboxamide, alkylenesulfonate, alkylenesulfonic acid, and thelike, a cyclic heteroatom-containing moiety, or an acyclicheteroatom-containing moiety, each of which may be optionallysubstituted; illustrative heteroatom-containing moieties includeoptionally substituted heteroalkyl, including methoxymethyl,ethoxyethyl, and the like, heterocyclyl, including piperidinyl, and thelike, alkyl and arylsulfonates, including methylsulfonate,4-chlorophenylsulfonate, and the like, alkoxy, including methoxy,ethoxy, and the like, amino, including methylamino, dimethylamino, andthe like, carbonyl derivatives, including alkyl and aryl carbonyl,alkylaminocarbonyl, alkoxycarbonyl, and the like, heteroalkenyl,including alkenylaminoalkyl, alkenyloxyalkyl, alkylaminoalkenyl,alkyloxyalkenyl, alkylideneaminoalkyl, and the like, heteroallyl,esters, amines, amides, phosphorus-oxygen, and phosphorus-sulfur bonds;and including heteroatom-containing moieties as described in U.S. Pat.No. 5,658,751 and PCT Publication No. WO 00/66664; the disclosures ofeach are herein incorporated in their entirety by reference;

t=0 or 1;

Z is a charge selected from 0 or 1;

R³, R⁹, and R¹⁰ are each independently selected from hydrogen, alkyl,including C₁₋₆ alkyl and C₂₋₆ alkyl, and arylcarbonyl;

n=0, 1, or 2; and

Q is a heterocycle, such as a pyridinium, a pyrimidinium, a quinolinium,or a purinium, each of which may be optionally substituted.

The term “alkyl” as used herein generally refers to a linear oroptionally branched hydrocarbon moiety comprising from 1 to about 12carbon atoms, illustratively including but not limited to methyl (Me),ethyl, propyl, butyl, dodecyl, 4-ethylpentyl, and the like.

The term “cycloalkyl” as used herein generally refers to a linear oroptionally branched hydrocarbon moiety, at least a portion of whichforms one or two rings, comprising from 3 to about 14 carbon atoms,illustratively including but not limited to cyclopropyl, cyclopentyl,cyclohexyl, 4-methylcyclohexyl, 2,3-dimethylcyclopentyl,3,5-dimethylcyclohexylethyl, and the like.

The term “aryl” as used herein generally refers to a cyclic aromaticmoiety, illustratively including but not limited to phenyl (Ph),naphthyl, furyl, thienyl, pyrrolo, pyrazolo, isoxazolyl, isothiazolyl,oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl,quinolinyl, isoquinolinyl, quinoxalinyl, quinazalinyl, and the like.

The term “optionally substituted” as used herein generally refers to theoptional replacement of one or more hydrogen atoms or lone electronpairs present on the parent group, including those present on carbon,nitrogen, oxygen, or sulfur atoms, with a substituent, such as halo;hydroxy; amino; nitro; thio; sulfonate; alkyl, cycloalkyl, haloalkyl,halocycloalkyl; alkoxy, cycloalkoxy, haloalkoxy; monoalkyl anddialkylamino; trialkylammonium; aminoalkyl; monoalkyl anddialkylaminoalkyl; trialkylammoniumalkyl; trialkylammoniumalkylthio;alkylthio; cycloalkylthio, cycloheteroalkylthio, nucleosidylthio; alkyl,haloalkyl, cycloalkyl, and arylcarbonyl; arylcarbonylthio,cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio; alkyl,haloalkyl, cycloalkyl, and arylcarbonyloxy; alkyl, haloalkyl,cycloalkyl, and arylsulfonyl; and carboxyl derivatives, such ascarboxylic acids, esters, and amides. It is appreciated that thereplacement of proximal hydrogen atoms, including geminal and vicinalhydrogens, may be such that the substituents replacing those proximalhydrogens are taken together to form a spiro ring or a fused ring,respectively.

It is appreciated that each of the above described terms may be used incombination in chemically relevant ways to refer to other moieties, suchas arylalkyl referring to an aryl group as defined herein linked to analkyl group as defined herein to form structures including, but notlimited to, benzyl, phenethyl, picolinyl, 3,5-dimethoxypicolin-4-yl, andthe like.

It is appreciated that the cyanine dye structures described herein maycontain chiral centers. In those cases, all stereoisomers are understoodto be included in the description of these cyanine dye structures,unless otherwise indicated. Such stereoisomers include pure opticallyactive isomers, racemic mixtures, and mixtures of diastereomerscontaining any relative amount of one or more stereoisomericconfigurations.

It is also appreciated that the cyanine dye structures described hereinmay contain geometric centers. In those cases, all geometric isomers areunderstood to be included in the description of the cyanine dyestructures, unless otherwise indicated. Such geometric isomers includecis, trans, E and Z isomers, either in pure form or in various mixturesof geometric configurations. It is also understood that depending uponthe nature of the double bond contained in the cyanine dye structures,such double bond isomers may interconvert between cis and trans, orbetween E and Z configurations depending upon the conditions, such assolvent composition, solvent polarity, ionic strength, and the like.

It is further appreciated that when the charge Z is greater than 0,several resonance structures of the compounds of Formula I may exist.Illustratively, the charge Z may be formally localized on the nitrogenatom as depicted in Formula I, or alternatively, the charge may belocalized on the heterocycle Q. Resonance structures of the chargedcompounds of Formula I may be depicted by rearranging the doublebond-single bond configuration of compounds of Formula I, such as theillustrative structures:

wherein

, R², R³, R⁹, R¹⁰, and Q, are as defined for Formula I, and t=1, Z=1,and n=1. The cyanine dye compounds described herein include any of theseveral possible resonance structures. It is understood that thelocation of the formal charge and, hence, the favored resonancestructure, is influenced by the nature of the moieties,

, R², R³, R⁹, R¹⁰, and Q.

It is also understood that when compounds of Formula I carry a netcharge, such as where Z is 1, or where there is present on the compoundsof Formula I a charged substituent, such as an ammonium group, or asulfonic acid group, these compounds of Formula I are accompanied by acounter ion. Any monovalent, divalent, or polyvalent counter ion isincluded in the description of the cyanine dye structures containedherein. Illustrative counter-ions include negatively charged counterions such as iodide, chloride, bromide, hydroxide, oxide, acetate,trifluoroacetate, monophosphate, diphosphate, triphosphate, and thelike, and positively charged counter ions such as lithium, sodium,potassium, cesium, ammonium, polyalkylammonium, and the like. Suchcounter ions may arise from the synthetic methods used, the purificationprotocol, or other ion exchange processes.

It is believed that the nature or type of counter ion does not appear toinfluence the functionality of the cyanine dyes described herein. It isappreciated that when the dyes described herein are dissolved insolvents or other media used to practice the PCR reaction mixtures,methods, and compositions described herein, the accompanying counter ionmay exchange with other counter ions that are present in the solvents orother media. Such additional counter ions may be solvent ions, salts,buffers, and/or metals.

It is appreciated that the group R² may be virtually any group thatarises from the nucleophilic reaction between the parent compound ofFormula I, where t=Z=0:

and a compound having the formula R²-L, wherein L is a suitable leavinggroup, and R² is as defined above. Illustratively, R² is an optionallysubstituted alkyl, acyl, aryl, sulfonic acid, or sulfonyl group, each ofwhich may be optionally substituted. Illustrative leaving groups Linclude, but are not limited to halides, such as chloride and bromide,acylates, such as acetate, formate, and trifluoroacetate, sulfonates,such as methylsulfonate, trifluoromethylsulfonate, and tolylsulfonate,sulfates, such as methylsulfate, and the like.

In one illustrative embodiment, Q is an heterocycle such as, but notlimited to:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ are eachindependently selected from the group consisting of hydrogen, halogen,amino, alkyl, haloalkyl, alkoxy, haloalkoxy, alkylsulfonyl,haloalkylsulfonyl, arylsulfonyl, formyl, alkylcarbonyl, arylcarbonyl,carboxylic acid derivatives, monoalkylamino, dialkylamino,trialkylammonium, dialkylaminoalkyl, trialkylammoniumalkyl,trialkylammoniumalkylthio, cycloalkyl, heteroalkyl, heterocycloalkyl,alkenyl, polyalkenyl, alkynyl, polyalkynyl, alkenylalkynyl, aryl,heteroaryl, alkoxy, alkylthio, arylthio, arylcarbonylthio,cycloheteroalkylcarbonylthio, dialkylaminoalkylcarbonylthio,dialkylamino, cycloalkylthio, cycloheteroalkylthio, nucleosidylthio,each of which may be optionally substituted, piperidino, piperazino,each of which may be optionally substituted with alkyl, amino, mono ordialkylaminoalkyl, trialkylammoniumalkyl, or may be optionallyquaternized on the nitrogen with an alkyl group.

In another illustrative embodiment, one of R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹²,R¹³, and R¹⁴ is an heteroatom-containing moiety, as described in U.S.Pat. No. 5,658,751. In another illustrative embodiment, one of R⁴, R⁵,R⁶, R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ is a reactive group, including butnot limited to halogens, hydroxy, alkoxides, amines, carboxylic acids,halides, alcohols, aldehydes, thiols, alkyl, and arylthiols, alkyl andarylsulfonyls, succinimidyl esters, ketones, and isothiocyanates thatmay be used to attach moieties to the dye core structure, illustrativelythrough the formation of carbon-carbon bonds, amines, amides, ethers,thioethers, disulfides, ketones, thioureas, and Schiff bases. In anotherillustrative embodiment, one of R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², R¹³, andR¹⁴ is a BRIDGE-DYE having the formula:

wherein

, X, R², t, Z, R³, R⁹, R¹⁰, Q, and n are as defined for Formula I, andBRIDGE is a single covalent bond, or a covalent linkage that is linearor branched, cyclic or heterocyclic, saturated or unsaturated, having1-16 non-hydrogen atoms such as carbon, nitrogen, phosphate, oxygen, andsulfur, such that the linkage contains any combination of alkyl, ether,thioether, amine, ester, or amide bonds; single, double, triple, oraromatic carbon-carbon bonds; phosphorus-oxygen, phosphorus-sulfur,nitrogen-nitrogen, or nitrogen-oxygen bonds; or aromatic orheteroaromatic bonds. It is appreciated that in some embodiments, thisdimeric structure is symmetrical about BRIDGE, and in other embodiments,this dimeric structure is unsymmetrical about BRIDGE, wherein forexample, any of,

, X, R², t, R³, R⁹, R¹⁰ and n are each independently selected in eachoccurrence on each side of BRIDGE.

Illustrative dyes for use in the present invention also include cyaninedyes of Formula I having a pyridinium or pyrimidinium core structurewherein X is oxygen or sulfur; the moiety Y represents anoptionally-substituted fused benzo, optionally-substituted fusednaphthaleno, optionally-substituted fused pyridino,optionally-substituted fused pyrimidino, optionally-substituted fusedquinolino, and the like; n=0 or 1; t=0 or 1; R² is alkyl, such as methyland ethyl, optionally substituted aryl, such as phenyl or tolyl, analkylenesulfonate, such as propylenesulfonic acid, or alkylsulfonyl,such as CH₃(CH₂)_(m)SO₂, where m is 0, 1, 2, or 3; and Q is anheterocycle selected from the group of structures consisting of:

wherein

R⁴ is hydrogen, alkoxy, including methoxy, ethoxy, propyloxy, and thelike; alkylthio, including methylthio, ethylthio, and the like;heterocyclylalkyl, including optionally substituted piperidinyl,pyrrolidinyl, piperazinyl, and the like; or heterocyclylalkyl includinga charged group, including 4,4-dimethylpiperazinium-1-yl, and the like;or a reactive group, including halo, hydroxy, alkoxy, thio, alkyl andarylthio, alkyl and arylsulfonyl, amino, formyl, alkyl and arylcarbonyl,carboxyl derivatives, and the like;

R⁵ is C₁₋₆ alkyl, including methyl, ethyl, butyl, sec-butyl, isobutyl,and the like; optionally substituted phenyl; or (CH₂)₃N⁺(Me)₃; and

R⁶, R⁷, and R⁸ are each independently hydrogen or methyl.

Illustrative dyes for use herein also include cyanine dyes of Formula Ihaving a pyridinium or pyrimidinium core structure wherein X is oxygenor sulfur; the moiety Y represents an optionally-substituted fusedbenzo, forming an optionally substituted benzoxazolium or benzthiazoliumring, or an optionally-substituted fused naphtho, forming an optionallysubstituted naphthoxazolium or naphthothiazolium ring; n=0 or 1; t=0 or1; R² is alkyl, such as methyl, aryl, such as phenyl or tolyl, analkylenesulfonate, such as propylenesulfonic acid, or alkylsulfonyl,such as CH₃(CH₂)_(m)SO₂, where m is 0, 1, 2, or 3; and Q is a4-pyridinium or 4-pyrimidinium heterocycle.

Illustrative dyes for use herein also include cyanine dyes useful in thePCR reaction mixtures, methods, and compositions described herein withquinolinium core structures, and generally described by Formula II:

wherein

the moiety Y represents an optionally-substituted fused mono orpolycyclic aromatic or nitrogen-containing heteroaromatic ring;

X is oxygen, sulfur, or a group selected from C(CH₃)₂, and NR¹, where R¹is hydrogen or C₁₋₆ alkyl;

R² is alkyl, including C₁₋₆ alkyl and C₂₋₆ alkyl, cycloalkyl, includingC₃₋₈ cycloalkyl, aryl, arylalkyl, an alkylenesulfonate, a cyclicheteroatom-containing moiety, or an acyclic heteroatom-containingmoiety, each of which may be optionally substituted;

t=0 or 1;

Z is a charge selected from 0 or 1;

R³, R⁹, and R¹⁰ are each independently selected from hydrogen and alkyl,including C₁₋₆ alkyl;

n=0, 1, or 2; and

R⁴, R⁵, R⁸, R¹¹, R¹², R¹³, and R¹⁴ are as described herein for FormulaI, providing that R⁴ is a moiety with a molecular weight of less thanabout 115, or illustratively a molecular weight of less than about 105.

Illustrative dyes for use in the present invention also include cyaninedyes of Formula II wherein the moiety Y represents anoptionally-substituted fused benzo, thereby forming a benzoxazolium orbenzthiazolium ring; X is oxygen or sulfur; n=0 or 1; t=0 or 1; R² ismethyl;

R⁴ is hydrogen, C₁₋₆ alkyl, including methyl, or optionally-substitutedphenyl;

R⁵ is C₁₋₆ alkyl, including methyl, or optionally-substituted phenyl;

R⁸ is hydrogen, and

R¹¹, R¹², R¹³, and R¹⁴ are hydrogen or alkoxy, including methoxy.

In other embodiments, dyes for use in the present invention alsoillustratively include cyanine dyes of Formula II wherein the moiety Yrepresents an optionally-substituted heterocycle, including1-methylpyrido and 3-bromo-1-methylpyrido; X is oxygen or sulfur; n=0 or1; t=z=0;

R⁴ is hydrogen or C₁₋₆ alkyl, including methyl;

R⁵ is C₁₋₆ alkyl, including methyl, optionally-substituted phenyl orheteroalkyl, including heteroalkyl having a charged group such as thegroup —(CH₂)₃N(Me)₃;

R⁸ is hydrogen; and

R¹¹, R¹², R¹³, and R¹⁴ are hydrogen, alkyl, including methyl, or alkoxy,including methoxy.

In another embodiment, two compounds of Formula I are taken together toform a dimer. The two compounds are linked to each other by replacingone of the substituents R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴, asdefined above, present on each of the compounds of Formula I with asingle divalent linker. Illustratively, two compounds of Formula I aretaken together to form a dimer, where the two R⁵ substituents present onthe two compounds of Formula I are replaced with a single divalentlinker. It is appreciated that both symmetrical and unsymmetrical dimersof Formula I compounds are contemplated herein. In the case ofunsymmetrical dimers of compounds of Formula I, it is understood thatsuch asymmetry may arise by forming dimers from compounds of Formula Ihaving different substitution patterns, or having different heterocyclesQ. Further, such asymmetry may arise by forming dimers from compounds ofFormula I where different substituents are replaced with the divalentlinker, such as illustratively replacing R⁵ on a first compound ofFormula I and replacing R⁸ on a second compound of Formula I with thedivalent linker.

In another embodiment, two compounds of Formula II are taken together toform a dimer. The two compounds are linked to each other by replacingone of the substituents R⁴, R⁵, R⁸, R¹¹, R¹², R¹³, and R¹⁴, as definedabove, present on each of the compounds of Formula II with a singledivalent linker. Illustratively, two compounds of Formula II are takentogether to form a dimer, where the two R⁵ substituents present on thetwo compounds of Formula II are replaced with a single divalent linker.It is appreciated that both symmetrical and unsymmetrical dimers ofFormula II compounds are contemplated herein. In the case ofunsymmetrical dimers of compounds of Formula II, it is understood thatsuch asymmetry may arise by forming dimers from compounds of Formula IIhaving different substitution patterns, or having different heterocyclesQ. Further, such asymmetry may arise by forming dimers from compounds ofFormula II where different substituents are replaced with the divalentlinker, such as illustratively replacing R⁵ on a first compound ofFormula II and replacing R⁸ on a second compound of Formula II with thedivalent linker.

The dimeric cyanine dye structures formed by compounds of Formula I mayalso be represented by Formula III:

wherein

the moieties Y and Y′ each represent an independently selectedoptionally-substituted fused mono or polycyclic aromatic ornitrogen-containing heteroaromatic ring;

X and X′ are each independently selected from oxygen, sulfur, selenium,tellurium, or a group selected from C(CH₃)₂, NR¹, or NR^(1′), where R¹and R^(1′) are each independently hydrogen or C₁₋₆ alkyl;

R² and R^(2′) are each independently selected from alkyl, including C₁₋₆alkyl, cycloalkyl, including C₃₋₈ cycloalkyl, aryl, arylalkyl, includingaryl(C₁₋₂ alkyl), a cyclic heteroatom-containing moiety, or an acyclicheteroatom-containing moiety, each of which may be optionallysubstituted;

t=0 or 1;

t′=0 or 1;

Z and Z′ are each a charge independently selected from 0 or 1;

R³, R⁹, R¹⁰, R^(3′), R^(9′), and R^(10′) are each independently selectedfrom hydrogen and alkyl, including C₁₋₆ alkyl;

n=0, 1, or 2;

n′=0, 1, or 2;

BRIDGE is a divalent linker comprising 2 to about 30 divalent unitsselected from alkylene, heteroalkylene, alkylamindiyl,alkylalkylammoniumdiyl, and the like, such as (CH₂)_(p),(CH₂)_(p)N⁺Me₂(CH₂)_(q), (CH₂)_(p)N⁺Me₂(CH₂)_(q)N⁺Me₂(CH₂)_(r), and thelike, where p, q, and r are each independently selected from 1, 2, and3; and

Q and Q′ are heterocycles, each independently selected from the group ofstructures consisting of:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R¹¹, R¹², R¹³, and R¹⁴ are in eachoccurrence in compounds of Formula III independently selected from thegroup consisting of hydrogen, halogen, amino, alkyl, haloalkyl, alkoxy,haloalkoxy, alkylsulfonyl, haloalkylsulfonyl, arylsulfonyl, formyl,alkylcarbonyl, arylcarbonyl, carboxylic acid derivatives,monoalkylamino, dialkylamino, trialkylammonium, dialkylaminoalkyl,trialkylammoniumalkyl, trialkylammoniumalkylthio, cycloalkyl,heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl, alkynyl,polyalkynyl, alkenylalkynyl, aryl, heteroaryl, alkoxy, alkylthio,arylthio, arylcarbonylthio, cycloheteroalkylcarbonylthio,dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio,cycloheteroalkylthio, nucleosidylthio, each of which may be optionallysubstituted, piperidino, piperazino, each of which may be optionallysubstituted with alkyl, amino, mono or dialkylaminoalkyl,trialkylammoniumalkyl, or may be optionally quaternized on the nitrogenwith an alkyl group.

Illustrative cyanine dyes useful in the present PCR reaction mixtures,methods, and compositions also include, but are not limited to, S5,PO-PRO™-1, BO-PRO™-1, SYTO® 43, SYTO® 44, SYTO® 45, SYTOX® Blue,POPO™-1, POPO™-3, BOBO™-1, BOBO™-3, and other dyes having the generalFormulae IV:

and various novel dyes presented in Example 1, and other dyes having thegeneral Formulae V:

wherein n is 0, 1, or 2; R² is alkyl, hydroxyalkyl, alkoxyalkyl,aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl,alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, and thelike; R⁵ is alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono ordialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate,alkylenecarboxamide, alkylenesulfonate, optionally substituted phenyl,and the like; X is oxygen or sulfur; A, A′, and B each represent one ormore independently selected optional substituents, such as alkyl, halo,amino, haloalkyl, alkoxy, haloalkoxy, alkyl and arylsulfonyl,haloalkylsulfonyl, alkyl and arylthio, formyl, alkyl and arylcarbonyl,carboxyl derivatives, mono and dialkylamino, trialkylammonium,dialkylaminoalkyl, trialkylammoniumalkyl, trialkylammoniumalkylthio,cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl,alkynyl, polyalkynyl, alkenylalkynyl, aryl, heteroaryl,arylcarbonylthio, cycloheteroalkylcarbonylthio,dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio,cycloheteroalkylthio, nucleosidylthio, or a heterocycle includingpyrrolidino, piperidino, piperazino, each of which may be optionallysubstituted with alkyl, amino, mono or dialkylaminoalkyl,trialkylammoniumalkyl or may be optionally quaternized on the nitrogenwith an alkyl group, and the like; and BRIDGE is a divalent linkerhaving the formula (CH₂)_(p)N⁺Me₂(CH₂)_(q), where p and q areindependently 2 or 3, which includes the divalent linker(CH₂)₃N⁺Me₂(CH₂)₃. It is understood that when these dyes have a netcharge, they are accompanied by one or more counter ions, such ascounter anions including halide, alkanoate, phosphate, and the like, andcounter cations including lithium, sodium, potassium, cesium, ammonium,and the like.

Other illustrative dyes for use herein include, but are not limited toYO-PRO®-1, TO-PRO®-1, SYTO® 11, SYTO® 13, SYTO® 15, SYTO® 16, SYTO® 20,SYTO® 23, TOTO™-3, YOYO®-3 (Molecular Probes, Inc.), GelStar® (CambrexBio Science Rockland Inc., Rockland, Me.), thiazole orange (Aldrich),and other dyes having the general Formulae VI:

wherein n is 0, 1, or 2; R is alkyl, hydroxyalkyl, alkoxyalkyl,aminoalkyl, mono and dialkylaminoalkyl, trialkylammoniumalkyl,alkylenecarboxylate, alkylenecarboxamide, alkylenesulfonate, and thelike; R⁵ is alkyl, hydroxyalkyl, alkoxyalkyl, aminoalkyl, mono ordialkylaminoalkyl, trialkylammoniumalkyl, alkylenecarboxylate,alkylenecarboxamide, alkylenesulfonate, optionally substituted phenyl,and the like; X is oxygen or sulfur; A, B, and B′ each represent one ormore independently selected optional substituents, such as alkyl, halo,amino, mono and dialkylamino, pyrrolidino, piperidino, piperazino,phenyl, hydroxy, alkoxy, thio, and alkylthio, trialkylammoniumalkylthio,cycloalkyl, heteroalkyl, heterocycloalkyl, alkenyl, polyalkenyl,alkynyl, polyalkynyl, alkenylalkynyl, aryl, heteroaryl,arylcarbonylthio, cycloheteroalkylcarbonylthio,dialkylaminoalkylcarbonylthio, dialkylamino, cycloalkylthio,cycloheteroalkylthio, nucleosidylthio, each of which may be optionallysubstituted with alkyl, amino, mono or dialkylaminoalkyl,trialkylammoniumalkyl, and the like; and BRIDGE is a divalent linkerhaving the formula (CH₂)_(p)N⁺Me₂(CH₂)_(q), where p and q areindependently 2 or 3, which includes the divalent linker(CH₂)₃N⁺Me₂(CH₂)₃. It is understood that when these dyes have a netcharge, they are accompanied by one or more counter ions, such ascounter anions including halide, alkanoate, phosphate, and the like, andcounter cations including lithium, sodium, potassium, cesium, ammonium,and the like.

Initial results have indicated that S5, PO-PRO™-1, JO-PRO™-1, BO-PRO™-1,G5, H5, S5, D6, E6, P6, R6, Y6, Z6, N7, O7, P7, Q7, R7, T7, V7, Z7, G8,L8, P8, T8, V8, W8, Z8, A9, C9, G9, 19, J9, K9, L9, M9, N9, O9, and P9are quite promising dyes for heteroduplex detection. There are severalsurprising characteristics of these dyes. First, they do notsignificantly inhibit PCR at 50% saturation. In fact, saturation levelsfairly close to 100% are compatible with PCR with most of these dyes.Secondly, although some of the dyes emit in the blue range, they arecompatible with use in the fluorescein channel of a variety of currentlyavailable instruments. Adjustment of the optics to better match theexcitation/emission spectra of these dyes may further improve theirsensitivity for use in quantitative or qualitative amplificationanalysis.

It is understood that the above cyanine dyes are illustrative, and othercyanine dyes may be useful in the presently-described methods.

Some quinolinium-based unsymmetrical cyanines such as, but not limitedto, SYBR® Green I, SYTOX® Green, SYTO® 14, SYTO® 21, SYTO® 24, SYTO® 25,TOTO™-1 and YOYO®-1 have not proven useful for heteroduplex detection orfor the detection of multiple products in a closed-tube system. When thedye is a monomer of a quinolinium-based cyanine, it is possible thatbulky substitutions on the carbon next to the nitrogen of thequinolonium ring (position equivalent to R⁴) interfere with the dye'sability to function in the methods of the present invention. Bulkysubstitutions are, for example, long-chain branchedhetero-atom-containing aliphatic or aromatic moieties substituted withbranched-chain aliphatic moieties that are larger than MW of about 105.This restriction, however, does not apply to any of the pyridinium orpyrimidinium cyanines mentioned earlier. In the case ofquinolinium-based cyanine dimers, the distance between the left andright ring systems, as defined by the divalent fragment:

also appears to affect functionality. Functionality may be determined byheteroduplex detection, as taught herein in Example 1. Other dyespreviously described as useful in real-time monitoring of PCR, such asSYBR® Gold, Pico Green, and ethidium bromide have also been shown to beineffective in heteroduplex detection in a closed-tube PCR system.

The dyes for use in the present invention may be used in a dye-basedmethod for SNP genotyping, requiring only two unlabeled oligonucleotideprimers and one well for each SNP genotype, and not requiring real-timePCR. A dsDNA dye is used such that heterozygotes are identified by thepresence of heteroduplexes that alter the shape of thepost-amplification melting curve. Different homozygous genotypes aredifferentiated by their Tm difference, or alternately by mixing a knownhomozygous DNA sample with the unknown and looking for heteroduplexes.Illustratively, PCR primer design is greatly simplified because veryshort amplicons can be used, preferably immediately flanking the SNP.Such short amplicons also amplify very efficiently, reduce the risk ofamplifying alternate targets, and allow very rapid thermal cycling.

The design of PCR primers is not an exact science, and often trial anderror is necessary. Although some rules for PCR primer design aregenerally accepted, the validity of these rules has not been tested.Because the effect of different genotypes on melting curves is greaterwith short amplicons, short amplicons are preferred (≦100 bp), and theshortest possible amplicons are often best (≦50 bp). Therefore, todesign primers for genotyping with dsDNA dyes, one illustratively startswith each flanking primer right next to the SNP position. That is, theamplicon length will be the length of primer 1, plus the length ofprimer 2, plus the length of the region that needs to be tested (thelength of an SNP is 1). For efficient amplification, the meltingtemperature (Tm) of the two primers should be nearly the same.Convenient Tms for primers may be 50 to 70 degrees C. Primers with thehighest Tm illustratively will allow the fastest thermal cycling, whileprimers with lower Tm are generally less expensive and produce theshortest amplicons, resulting in greater genotyping differences. Primerlengths between 12 and 30 bases are usually used. Illustratively, eachprimer is built away from the SNP until the calculated Tm is closest tothe desired Tm. Methods for Tm calculation are well known in the art(e.g. Clin. Chem. 2001; 47:1956-61). In general, the primer lengths willnot be the same when the Tms are matched as closely as possible. Forexample, the primer lengths used in the Factor V SNP assay (FIG. 1) are17 and 24 bases long both with a calculated matched Tm near 62° C.

Thermal cycling parameters for amplification can be very short becauselittle primer extension is required for such short amplicons. After aninitial denaturation of genomic DNA before thermal cycling, denaturationand annealing temperatures do not need to be held, and the extensiontime can be 10 s or less. It is even possible to reduce the programmedextension time to zero, allowing each cycle to be performed in less than20 s. Alternately, an extension time of 1 s can be used. Because theamplicon is so short, large amounts of polymerase are not required (<0.6Units per 10 μl may be used).

One will appreciate, however, that the present invention is not limitedto the PCR parameters explicitly disclosed herein. For instance, thoseskilled in the art will appreciate that, in certain embodiments, certainPCR parameters can vary depending on the specific reagents included inthe reaction mixture. Thus, reference to a specific concentration,temperature, or time parameter should not be construed to necessarily belimited to the recited value thereof. Furthermore, as a PCR reaction isheated to the denaturation temperature, dsDNA in the sample can bemelted to single strands. This denaturation can be observed, in someembodiments, as a drop in the fluorescence of a dsDNA dye present in thesample.

Thus, the following illustrative steps may be followed for SNPgenotyping according to the present invention:

1. Choose a target Tm and start with the 3′-end of each primer rightnext to the SNP position. Optionally, one primer may be shifted slightlyaway from the SNP position to avoid 3′ complementarity between primersto decrease the risk of primer dimer formation.

2. Design each primer outward until the calculated Tm is as close aspossible to the target Tm.

3. Rapidly thermal cycle the sample in the presence of PCR reagents anda dsDNA dye that allows heteroduplex detection.

4. Form heteroduplexes by rapid cooling at a rate of at least −0.1°C./s, preferably at least −2° C./s, and most preferably at least −5°C./s after denaturation.

5. Heat at 0.1 to 0.5° C./s and acquire a melting curve.

6. If the amplification fails, move the 3′-end of one of the primers out1 base and repeat all steps until successful.

In an illustrated example, all heterozygotes can be detected by theeffect of the heteroduplexes on the melting curve (FIG. 4). In addition,4 out of 6 homozygous differences (A vs C, A vs G, C vs T, and G vs T)are very easily distinguished by Tm shifts (FIG. 4, arrows). However, todistinguish A vs T homozygotes or C vs G homozygotes, high resolutionmelting is often necessary, and in some cases, homozygotes cannot bedifferentiated even with the high resolution melting currentlyavailable. When the frequency of SNPs in the human genome is considered,in 84% of SNPs it is easy to distinguish the homozygotes (A vs C, A vsG, C vs T, and G vs T), while 16% are more difficult (A vs T and C vsG). Indeed, in 4% of cases (one quarter of 16%), stability calculationsusing nearest neighbor analysis indicates identical stabilities becauseof symmetry of neighboring bases. Exact frequencies are given in Table 1where SNPs are classified according to which homoduplexes andheteroduplexes are produced. In the cases where it is difficult todifferentiate homozygotes, an unlabeled probe may be preferred forcomplete, robust genotyping.

TABLE 1^(a) SNP Homoduplex Heteroduplex Heterozygote Matches MismatchesClass (frequency)^(b) (# of Tms) (# of Tms) 1 C vs T or G vs A C::G andA::T C::A and T::G (0.675) (2) (2 or 1)^(c) 2 C vs A or G vs T C::G andA::T C::T and A::G (0.169) (2) (2 or 1)^(c) 3 C vs G C::G C::C and G::G(0.086) (2 or 1)^(c) (2) 4 T vs A A::T T::T and A::A (0.070) (2 or1)^(c) (2) ^(a)SNP heterozygotes are specified with the alternate basesseparated by “vs”, for example C vs T indicates that one allele has a Cand the other a T at the same position on the same strand. There is nobias for one allele over the other, that is, C vs T is equivalent to Tvs C. Base pairing (whether matched or mismatched) is indicated by adouble colon and is not directional. That is, C::G indicates a C::G basepair without specifying which base is on which strand. ^(b)The human SNPfrequencies were taken from the Kwok data set as reported in Venter JC,et al. The sequence of the human genome. Science 2001; 291: 1304-51).^(c)The number of predicted thermodynamic duplexes depends on thenearest neighbor symmetry around the base change. One quarter of time,nearest neighbor symmetry is expected, that is, the position of the basechange will be flanked on each side by complementary bases. For example,if a C vs G SNP is flanked by an A and a T on the same strand, nearestneighbor symmetry occurs and only one homoduplex Tm is expected.

Alternatively, in the cases where differentiation of homozygotes isdifficult, a sample of a known homozygous genotype may be mixed inroughly equal amounts with the unknown genotype either before or afteramplification. The mixture is amplified (if not previously amplified),denatured, and melted. If the genotypes are the same, the melting curveof the mixture will be the same as the melting curve of the knownhomozygous genotype. If the genotypes are different, heteroduplexes willbe produced and identified by an altered shape of the melting curve.

Illustratively, small amplicons or unlabeled probes may be used whengenotyping for known sequence variants, while large amplicons may bepreferred when scanning for unknown variants. Multiplexing of ampliconsmay also be used. For example, if a smaller segment within a largeamplicon is known to carry a sequence variant of interest, then both thesmaller segment and the full-length amplicon can be amplified and meltedin one reaction. The melting data from the large amplicon will providemutation scanning information, while the melting data from the smallersegment may provide genotyping information. Amplification of both thelarger and the smaller amplicon can be performed simultaneously, or by abiphasic PCR which amplifies the larger amplicon in the first phase, andthe smaller amplicon(s) in the second phase, or vice versa. Thisbiphasic PCR can be achieved by designing the Tm and amount of each ofthe primers in a way that, by adjusting the annealing temperatures ofthe two phases, preferential amplification of the different ampliconsoccur. When the signal from the larger amplicon is expected to overwhelmor mask the signal from the shorter amplicon, this biphasic techniquecan be used to adjust the final amount of each of the amplicons tocircumvent such a problem.

Simultaneous scanning and genotyping can also be performed in a singlePCR when one or more unlabeled probes are included. Both the productmelting transition and the probe melting transitions are analyzed.Illustratively, the full length PCR product melting transition is firstanalyzed to detect any heteroduplexes present. Any sequence differencewithin the amplicon should be detected by this scanning analysis. If asequence variant is detected by scanning, the melting transition(s) ofthe unlabeled probe(s) present reveal the genotype for each probe'slocus. Because the probes are smaller than the whole PCR product,genotyping with unlabeled probes is more specific than whole amplicongenotyping, and all SNP changes at the probe locus can be genotyped.

Example 1 Dye Synthesis

Unsymmetrical cyanine dyes can be prepared by a general method thatattaches the benzazolium portion of the molecule to the pyridinium (orquinolinium, pyrimidinium, purinium) portion through one or more“—C(R)═” groups. As described in U.S. Pat. No. 5,436,134 and referencescited therein, the number of “—C(R)═” groups is determined by thespecific synthetic reagents used in the synthesis. In the synthesis ofmonomethine dyes (R³═H, n=0) such as dye S5, a combination of reagentsis used in which the methine carbon atom results from either A on thebenzazolium salt or B on the pyridinium salt being methyl and the otherof A or B being a reactive leaving group that is typically methylthio,methylsulfonyl, or chloro, but which can be any leaving group thatprovides sufficient reactivity to complete the reaction. One possibleway to prepare dye S5 and other similar dyes may be as follows:

The starting material, Compound 1 is prepared by heating4-methyl-2-pyridinone (Aldrich) to reflux with copper powder, potassiumcarbonate and iodobenzene for 48 hours. The reaction is cooled to roomtemperature, partitioned between water and ethyl acetate, filtered, andthe organic layer is dried over magnesium sulfate. The crude product ispurified on a silica gel column, eluting with 1:1 ethyl acetate/hexanesto yield Compound 1.

Another starting material, Compound 2, is prepared by adding2-(methylthio)benzoxazole to methyl iodide in DMF and heating in asealed tube at 150° C. for one hour to obtain Compound 2, as the iodidesalt.

A mixture of Compound 1, phosphorous oxychloride, and a catalytic amountof DMF in methylene chloride is heated to reflux for 24 hours. Themixture is cooled to room temperature and another volume of methylenechloride is added, followed by Compound 2 and one equivalent oftriethylamine. The mixture is stirred at room temperature for 6 hours. Asolid is separated by filtration and purified using a silica gel columneluting with a mixture of ethyl acetate/chloroform/methanol. Thepurified compound is then redissolved in methanol and added to an excessof sodium iodide in water. Compound 3 is isolated by filtration as theiodide salt and dried in vacuo.

Compound 3 is then mixed with 1-methylpiperazine in 1,2-dichloroethaneand heated at 55° C. for 2 hours. The resulting product (Compound 4) isthen quaternized by adding an excess of methyl iodide and Proton Sponge(Aldrich), and is expected to yield dye S5 (Compound 5) as the diiodidesalt.

Additionally, certain embodiments of dyes having the followingpyrimidinium core structure have been prepared:

wherein Y, X, R², R³, and R⁵ are as defined herein for Formula I, and Bis as defined in Formulae V.

While there are many ways of preparing dyes having this formula, onemethod is as follows:

where compounds 6 are commercially available, or may be prepared byconventional methods. Compounds 7a are prepared by alkylation of 6 atN(3) using alkylating agents such as alkyl halides, alkylsulfates, andthe like, under neutral or basic conditions, including alkyllithiums,aromatic and aliphatic amines, K₂CO₃, and the like. Similarly, compounds7a are prepared by arylation of 6 at N(3) by aromatic coupling reactionsof aromatic halides, boronates, and the like, which are catalyzed bymetal compounds, such as copper, palladium, platinum, and likecatalysts. Compounds 7b are prepared from compounds 7a by conventionaloxidation, such as reactions using hydrogen peroxide, peroxy acids,including m-CPBA, and the like. In some cases, compounds 7a or compounds7b are commercially available. Compounds 8 are commercially available orare prepared illustratively by condensation of appropriately substituted1,3-diones and ureas or thioureas. Further, compounds 8 having a thiol,alkoxy, or primary/secondary amine at C(2) may be modifiedillustratively by reacting with alkylhalides, alkoxyhalides, or anyreactant with a good leaving group under neutral conditions. Compounds 9may be prepared by reacting compounds 7 and compounds 8 under basicconditions, as described herein.

Exemplary compounds having this formula were prepared as hereindescribed, purified by HPLC using triethylamine-ammonium acetate as themobile phase, and isolated as their corresponding acetate salts. Theseexemplary compounds are illustrated in Table 2.

TABLE 2 Dye

X R² R⁵ B G5

S Ph

H H5

S Me

H I5

S

H K5

S Me

H L5

S —

H D6

S Me

H E6

O Me

H P6

S

H R6

S Me

H Y6

O Me

H Z6

S Me

H F7

S Me Ph 2-[4-(N,N-dimethyl piperazine)] 6-Me N7

S Me Ph

O7

S Me Ph 2-[S-(C(O)-4-pyridine)] 6-Me P7

S Me Ph 2-[S-(1-bicyclo [2.2.1]heptane)] 6-Me Q7

S Me Ph 2-[S-(4-(N-methyl- piperadine))] 6-Me R7

S Me Ph

S7

S Me Ph 2-[S-(C(O)-4-pyridine)] 6-Me T7

S Me Ph 2-[S-(C(O)-4-PhNO₂)] 6-Me U7

S Me Ph 2-SMe 6-Me V7

S Me Ph

W7

S None Ph

X7

S Me Ph

Z7

S Me Ph 2-SCH₃ 6-Me C8

S Me Ph 2-NH₂ 6-Me E8

S Me Ph 2-OH 6-Me G8

S Me Ph 2-SCH₃ 6-Me K8

S

Ph 2-SCH₃ 6-Me L8

S None Ph 2-SCH₃ 6-Me M8

S Me Ph [S-(2-pyrimidine)] 6-Me N8

S Me Ph 2-SMe 6-Ph O8

S Me R³═C(O)PH

H P8

S Me Ph 2-S-benzyl 6-Me T8

S Me Ph 2-[S-(C(O)-Ph)] 6-Me V8

S Me Ph 2-SCH₃ 6-Me W8

S Ph Ph 2-SCH₃ 6-Me X8

S Me Ph 2-SCH₃ 6-Me Z8

S Me 1-Naphthyl 2-SCH₃ 6-Me A9

S Me Ph 2-[S-(5′-deoxyAdenosine)] 6-Me C9

S Me Ph

G9

S

Ph 2-SCH₃ 6-Me I9

S Me Ph 2-S-C(O)-CH₂-NMe₂ 6-Me J9

S Me Ph 2-[S-(C(O)-4-(N- methylpiperazine))] 6-Me K9

S Me Ph 2-[S-(C(O)-4-morpholine)] 6-Me L9

S Me Ph 2-[S-(C(O)-4-(N,N- dimethylaniline))] 6-Me M9

S Me Ph 2-[S-(C(O)-2-pyrazine)] 6-Me N9

S Me Ph 2-[S-(C(O)-6- benzopyrazine)] 6-Me O9

S Me Ph 2-[S-(C(O)-5-(1-methyl- 1,2,3-benzotriazole))] 6-Me P9

S Me Ph 2-[S-(C(O)-C₆F₅)] 6-Me Q9

O None Ph 2-SCH3 6-Me R9

O None Ph

A10

S Me Ph

Compound D6 was prepared by first reacting 4-methylpyrimidine with(3-bromopropyl)trimethylammonium bromide in acetonitrile at reflux Theresulting product (compound A6) in acetonitrile was reacted with3-methyl-2-methylsulfonylbenzothiazolium iodide (available from Aldrich)in the presence of anhydrous pyridine and triethylamine, or inchloroform:methanol (10:1) and an excess of triethylamine. The reactionwas carried out either at reflux, or at room temperature.

Compound E6 was prepared according to the general procedure used toprepare compound D6 from 3-methyl-2-methylsulfonylbenzoxazolium iodide(prepared by reacting 2-methylsulfonylbenzoxazole with dimethylsulfate)and compound A6.

Compound G5 was prepared according to the general procedure used toprepare compound D6 from 2-methylthio-3-phenylbenzothiazolium (Aldrich)and compound A6.

Compound H5 was prepared according to the general procedure used toprepare compound D6 from5-difluoromethylsulfonyl-3-methyl-2-methylthiobenzothiazoliummethylsulfate (prepared by reacting5-difluoromethylsulfonyl-2-methylthiobenzothiazole, available fromAldrich, with dimethylsulfate) and compound A6.

Compound P6 was prepared according to the general procedure used toprepare compound D6 from5-chloro-2-(methylthio)-3-(3-sulfopropyl)-benzothiazolium hydroxide(Aldrich) and compound A6.

Compound R6 was prepared according to the general procedure used toprepare compound D6 from 6-amino-3-methyl-2-methylthiobenzothiazoliummethylsulfate (prepared by reacting 6-amino-2-methylthiobenzothiazole,available from Aldrich, with dimethylsulfate) and compound A6.

Compound Y6 was prepared according to the general procedure used toprepare compound D6 from3-methyl-2-methylsulfonylnaphtho[1,2-d]oxazolium methylsulfate (preparedby reacting 2-methylsulfonylnaphtho[1,2-d]oxazole, available from ChemBridge Product List, San Diego, Calif., with dimethylsulfate) andcompound A6.

Compound Z6 was prepared according to the general procedure used toprepare compound D6 from3-methyl-2-methylsulfonylnaphtho[1,2-d]thiazolium methylsulfate(prepared by reacting 2-methylsulfonylnaphtho[1,2-d]thiazole, availablefrom Specs, Rijswijk, The Netherlands, with dimethylsulfate) andcompound A6.

Compound G8 was prepared by heating a solution of N-phenylthiourea and2,4-pentanedione in HCl/EtOH at reflux. The resulting pyrimidinthionewas reacted with 3-methyl-2-methylsulfonylbenzothiazolium iodide in thepresence of triethylamine in chloroform/methanol (10:1) at refluxovernight to give compound G8.

Compounds G5, I5, K5, L5, F7, N7, O7, P7, Q7, R7, S7, T7, U7, V7, W7,X7, Z7, C8, E8, K8, L8, M8, N8, 08, P8, T8, V8, W8, X8, Z8, A9, C9, G9,19, J9, K9, L9, M9, N9, O9, P9, Q9, R9, and A10 may be prepared bysimilar methods described above. These dyes are dsDNA binding dyes whosefluorescence changes upon binding to dsDNA. It is expected that many ofthese dyes would be useful for detection of heteroduplexes.

The pyrimidinium-based cyanine dyes described herein, illustratively G5,H5, I5, K5, L5, D6, E6, P6, R6, Y6, Z6, F7, N7, O7, P7, Q7, R7, S7, T7,U7, V7, W7, X7, Z7, C8, E8, G8, K8, L8, M8, N8, 08, P8, T8, V8, W8, X8,Z8, A9, C9, G9, 19, J9, K9, L9, M9, N9, O9, P9, Q9, R9, and A10 arenovel. The results of using some of these dyes in the detection ofheteroduplexes are summarized in Table 3. In general, dyes that inhibitPCR at levels below 50% saturation do not detect heterozygotes well. PCRmethods and heteroduplex detection are as discussed in the followingexamples.

TABLE 3 Maximum PCR Dye Ex/Em¹ compatible % Sat² % Het³ G5 442-458/475100% 20.0% H5 444/459 100% 22.5% S5 450/469 >99% 20.5% D6 457/471 92%23.3% E6 425/454 >99% 15.0% P6 464/490 100% 21.0% R6 453/470 >90% 15.0%Y6 439/477-515 100% 21.0% Z6 469/494-526 100% 13.4% N7 458/474 >100%22.0% O7 **⁴ >70% 21.2% P7 **⁴ >70% 21.5% Q7 453/471 >70% 20.7% T7453/471 >70% 21.2% G8 453/471 >100% 19.7% L8 470-490/490-520 >70% 2.3%P8 453/471 >70% 17.7% T8 453/471 >70% 24.0% V8 469/494-526 >70% 21.4% W8453/471 >70% 27.5% Z8 453/471 >70% 22.7% A9 **⁴ 100% 23.0% I9 **⁴ 100%20.9% J9 **⁴ 100% 22.4% K9 **⁴ 100% 22.3% L9 **⁴ 100% 21.3% ¹Excitationmaxima (Ex) and emission maxima (Em) obtained in a fluorimeter using 2.5μM bp (100 ng/60 μl) of dsDNA and dye at maximum PCR compatibleconcentration in PCR buffer (3 mM MgCl₂, 50 mM Tris, pH 8.3, 200 μM eachdNTP, 500 μg/ml BSA). Some dyes have a range due to the broad emissionor excitation peak. ²Maximum amount of dye that can be present in a PCRmixture that allows amplification without significant inhibition,expressed as percentage of fluorescence compared to fluorescence of thesame dye at saturating concentration, i.e. the concentration thatprovides the highest fluorescence intensity possible, all in thepresence of 15 μM bp DNA (100 ng of dsDNA/10 μl) and PCR buffer.³Percentage peak area of the heteroduplex signature peak as measuredwith 420-490 nm excitation and 450-530 nm detection optics, using thedel F508 heterozygote melting curve obtained at a heating ramp of 0.3°C./s. The amplicon used in this set of experiments were 57 bp longgenerated by primers GGCACCATTAAAGAAAATAT (SEQ ID NO. 1) andTCTGTATCTATATTCATCATAGG (SEQ ID NO. 24) Maximum % obtained was recorded⁴Spectral data not available.

Example 2 PCR Protocol

Labeled and unlabeled oligonucleotides were obtained from IT Biochem(Salt Lake City, Utah), Qiagen Operon (Alameda, Calif.), or Synthegen(Houston, Tex.). PCR was performed in 10 μl volumes in a LightCycler®(Roche Applied Systems, Indianapolis, Ind.) with programmed transitionsof 20° C./s unless otherwise indicated. The amplification mixtureincluded 50 ng of genomic DNA as template, 200 μM of each dNTP, 3 mMMgCl₂, 100 mM 2-amino-2-methyl-1, 3-propanediol, pH 8.8, 0.04 U/l Taqpolymerase (Roche), 500 μg/ml bovine serum albumin, and 0.5 μM of eachprimer unless indicated otherwise. Genotyped human genomic DNA wasobtained from prior studies (Gundry C N, et al., Genetic Testing, 1999;3:365-70; Herrmann M, et al., Clin Chem 2000; 46:425-8) or from CoriellCell Repositories (Camden, N.J.). Dye S5 was included in the PCRreaction at 10 μM unless otherwise indicated. When SYBR® Green I wasused as the indicator, a 1:10,000 final dilution from the MolecularProbes stock was used. The dye is added before PCR, amplificationperformed, and the melting transition of the amplicon is monitored onthe LightCycler® or by high resolution melting analysis. Differenthomozygotes are distinguished by amplicon melting temperature (Tm).Heterozygotes are identified by low temperature melting ofheteroduplexes that broaden the overall melting transition. Meltinganalysis requires about 1 min and no sample processing is needed afterPCR.

To study the sensitivity of dye S5, SYBR® Green I, and other dsDNAbinding dyes, polymorphisms in Factor V Leiden, cystic fibrosis(F508del, F508C, 1507del, 1506V), and HTR2A (T102C) genes were analyzed.In addition, engineered plasmids were used to systematically study allpossible single base changes. Heteroduplexes produced by amplificationof heterozygous DNA were best detected by rapid cooling (at least −2°C./s) of denatured products, followed by rapid heating during meltinganalysis (0.2 to 0.4° C./s). All heterozygotes were distinguished fromhomozygotes by a broader melting transition. Different homozygotes couldoften be distinguished by their Tm. Homozygous G to C base changes couldnot reproducibly be distinguished, even with high resolution analysis,without mixing homozygotes. The amplicons varied in length from 44 to331 bp.

While the dyes S5, D6, Z6 and N7 are used in the Examples providedherein, it is understood that other dyes according to this invention maybe used.

Example 3 Melting Curve Analysis

Melting analysis was performed on the LightCycler® immediately aftercycling, or subsequently on either the high-resolution meltinginstrument HR-1 (Idaho Technology, Salt Lake City, Utah) or theLightTyper® (Roche Applied Systems, Indianapolis, Ind.). However, it isunderstood that melting curve analysis may be performed in the absenceof amplification. When the LightCycler® was used, the samples were firstheated to 94° C., cooled to 60° C. at a program setting of −20° C./s,then melted at 0.2° C./s with continuous acquisition of fluorescence.For melting in one of the other instruments, the samples were firstamplified in the LightCycler®, then heated momentarily in theLightCycler® to 94° C. and rapidly cooled (program setting of −20° C./s)to 40° C., unless stated otherwise. The LightCycler® capillaries werethen transferred one at a time to the high-resolution instrument andheated at 0.3° C./s unless otherwise stated. The HR-1 is a single sampleinstrument that surrounds one LightCycler® capillary with an aluminumcylinder. The system is heated by Joule heating through a coil woundaround the outside of the cylinder. Sample temperature is monitored witha thermocouple also placed within the cylinder and converted to a 24-bitdigital signal. Fluorescence is monitored by epi-illumination of thecapillary tip (Wittwer C T, et al., BioTechniques 1997; 22:176-81) thatis positioned at the bottom of the cylinder and also converted to a24-bit signal (it is noted that some of the examples used an earlier16-bit HR-1 prototype). Approximately 50 data points are acquired forevery ° C. Standard optics were used on all instruments unless otherwisenoted.

In some cases it is advantageous not to denature the product after PCRbefore melting curve acquisition. For example, when the goal is to typethe number of repeat sequences (e.g. STRs, VNTRs), amplification may bestopped at the extension step during the exponential phase of thereaction before plateau, and then melting analysis is performed. Thisway, homoduplex extension products can be analyzed. In repeat typing,homoduplex products can be more informative than heteroduplex products,especially since many different heteroduplex products may form fromdifferent alignment of the repeats. In some cases, it may be helpful toobtain both a homoduplex melting curve (without prior denaturation) anda heteroduplex melting curve (with denaturation and the formation of allpossible duplex combinations). The difference between these two meltingcurves gives a measure of the extent of heteroduplexes that can beformed, using the same sample as the “homoduplex control”.

Melting data were analyzed with custom software written in LabView.Fluorescence vs temperature plots were normalized between 0 and 100percent by first defining linear baselines before and after the meltingtransition of each sample. Within each sample, the fluorescence of eachacquisition was calculated as the percent fluorescence between the topand bottom baselines at the acquisition temperature. In some cases,derivative melting curve plots were calculated from the Savitsky-Golaypolynomials at each point (Press W H, et al., eds. Numerical recipes inC, 2^(nd) ed. New York: Cambridge University Press, 1992:650-5).Savitsky-Golay analysis used a second-degree polynomial and a datawindow including all points within a 1° C. interval. Peak areas andmelting temperatures were obtained by using non-linear least squaresregression to fit multiple Gaussians. In some cases, the X-axis for eachnormalized melting curve was translated so that the tracings overlappedwithin a certain fluorescence range. This “temperature shifting”corrects for any minor inter-run temperature variation and increases theability to distinguish heterozygotes from homozygotes. The differencebetween genotypes can also be magnified by plotting the fluorescencedifference between genotypes at each temperature.

Example 4 Single Nucleotide Polymorphism Genotyping with Dye S5:Genotyping the Factor V Leiden Mutation

A 43 bp amplicon was formed from primers 18 and 24 bases in length,immediately flanking the location of the factor V Leiden mutation. Bothprimers had an estimated Tm of 62° C. The samples were cycled 35 timeswith the following protocol: 94° C. with no hold, 60° C. with no hold,and 72° C. with a 10 s hold. After amplification, the samples wereheated momentarily in the LightCycler® to 94° C., cooled rapidly(program setting of −20° C./s) to 60° C., and PCR products melted at0.2° C./s with continuous fluorescence acquisition.

Derivative melting curves of PCR products amplified from differentgenotypes at the Leiden locus of the factor V gene are shown in FIG. 1.Dye S5 was used for fluorescent monitoring of the melting transitionbetween double- and single-stranded products. The Leiden mutation islocated 19 bases from one end of the amplicon. Results from tenhomozygous wild type, two heterozygous, and one homozygous Leidengenotypes are shown. The amplicon melting temperature of the homozygousmutant is about 1° C. less than the homozygous wild type meltingtemperature. Heterozygous samples show a secondary, low temperaturemelting transition attributable to heteroduplex formation. A similarexperiment using SYBR® Green I failed to detect this secondary meltingtransition in heterozygotes (data not shown).

The effects of cooling rate and heating rate were studied usingheterozygous factor V Leiden DNA on the LightCycler®. To study theeffect of cooling rate, the samples were amplified as above, heated to85° C., and then cooled from 85° C. to 60° C. at rate of −20, −2, −1,−0.5, or −0.1° C./s, followed by a constant heating rate of 0.2° C./sfor melting curve acquisition. Rapid cooling was necessary forsignificant heteroduplex formation (FIG. 2). Heteroduplexes were notobserved when the cooling rate was −0.1° C./s or slower. The greatestheteroduplex formation occurred when capillary samples were rapidlytransferred from boiling water to ice water (data not shown). Withcooling on the LightCycler®, heteroduplex formation appeared to plateauat programmed rates faster than −5° C./s (FIG. 2). However, measurementof actual sample temperatures showed that the cooling rate increasedonly slightly with programmed rates faster than −5° C./s: when theinstrument was programmed to cool at −20° C./s, the actual rate wasabout −6° C./s.

The effect of heating rate was studied by cooling at a programmed rateof −20° C./s, followed by melting at 0.05, 0.1, 0.3, or 0.5° C./s. Therelative percentage of observed heteroduplexes was greater with higherheating rates (FIG. 3). The apparent Tm also shifts to highertemperatures as the rate increases and the melting process deviates morefrom equilibrium (Gundry C N, et al., Genetic Testing, 1999; 3:365-70).

Example 5 Systematic Study of SNP Genotyping with Plasmids

Engineered plasmids were used for systematic study of melting curvegenotyping of all possible single base changes. The plasmids (DNAToolbox, Cambrex Bio Science Rockland Inc.) contained either A, C, G, orT at a defined position amid 50% GC content (Highsmith W E, et al.,Electrophoresis 1999; 20:1186-94). The four plasmids were either usedalone to simulate homozygous genotypes, or in binary combinations toconstruct “heterozygotes”. Primers were TCTGCTCTGCGGCTTTCT (SEQ ID NO.50) and CGAAGCAGTAAAAGCTCTTGGAT (SEQ ID NO. 51) and produced a 50 bpamplicon around the polymorphic position. The DNA templates were used at10⁶ copies and PCR was performed with 35 cycles of 85° C. with no holdand 55° C. for 1 sec in the presence of 20 uM D6. The HR-1high-resolution melting instrument was used for melting analysis.

The normalized melting curves of the four homozygotes (FIG. 4A) and sixheterozygotes (FIG. 4B) are shown. It is easy to distinguish thehomozygotes from the heterozygotes, because the heterozygotes have anextended melting transition that arises from the presence ofheteroduplexes. All homozygotes melt in a single transition (FIG. 4A)and the order of melting is correctly predicted by nearest neighborcalculations as A/A<T/T<C/C<G/G (SantaLucia J., Jr, Biochemistry 1996;35:3555-62). Heterozygotes result in more complex melting curves arisingfrom contributions of two homoduplexes and two heteroduplexes (FIG. 4B).Each heterozygote traces a unique melting curve path according to thefour duplex Tms. The order of melting is again according to nearestneighbor calculations (A/T<A/C<C/T<A/G<G/T<C/G) using the average of thetwo homoduplex Tms. The six heterozygote curves merge at hightemperatures into three traces, predicted by the highest meltinghomoduplex present (T/T for the A/T heterozygote, C/C for the A/C andC/T heterozygotes, and G/G for the A/G, G/T, and C/G heterozygotes). Allgenotypes can be distinguished from each other with high-resolutionmelting analysis.

Example 6 Genotyping of the Cystic Fibrosis Gene with Labeled Primers:Dye S5 or SYBR® Green I

KlenTaq1 polymerase (0.04 U/μl, AB Peptides, St. Louis, Mo.), 88 ng ofTaqStart antibody (ClonTech, Palo Alto, Calif.), and 50 mM Tris, pH 8.3were used in PCR instead of Taq polymerase and 2-amino-2-methyl-1,3-propanediol. A 44 bp fragment was amplified with the primersggcaccattaaagaaaatat (SEQ ID NO. 1) and TCATCATAGGAAACACCA (SEQ ID NO.2). The first primer was either 5′-labeled with Oregon Green, or thereaction was performed in the presence of SYBR® Green I or S5. Theprimers flank the mutational hot spot containing the F508del, I507del,and F508C variants. PCR was performed through 40 cycles of 85° C. and58° C. (0 s holds). Six samples were monitored during melting curveacquisition on the LightCycler®.

Derivative melting curves of PCR products amplified from differentgenotypes at the 1507/F508 region of the cystic fibrosis gene are shownin FIGS. 5B-D. The PCR products were 41 or 44 bases long (FIG. 5A).Either a 5′-labeled primer (FIG. 5B), dye S5 (FIG. 5C), or SYBR® Green I(FIG. 5D) was used for fluorescent monitoring of the melting transitionbetween double and single stranded products. Results from two homozygousand three heterozygous genotypes are shown.

The duplex stability of the different genotypes follows theoreticalcalculations (von Ahsen N, et al., Clin Chem 2001; 47:1956-61), withF508del˜I507del<Wild type<F508C. Except for F508del and I507del, thegenotypes are distinguishable by the Tms of their major transitions. Thestandard deviation of the Tm of 10 replicate wild type samples was 0.12°C. when melted on the LightCycler®. When melted on the high-resolutioninstrument, the standard deviation of the Tm of the same 10 samples was0.04° C.

When a heterozygous sample is amplified by PCR, two homoduplex and twoheteroduplex products are expected (Nataraj A J, et al., Electrophoresis1999; 20:1177-85). However, when SYBR® Green I was used as thefluorescent indicator, only a single melting peak was apparent for eachgenotype (FIG. 5D). In contrast, when labeled primers or dye S5 are usedunder the same conditions, two clearly defined peaks appeared (FIGS. 5Band 5C). The lower temperature peak is always smaller than the highertemperature peak, and presumably indicates the melting transition of oneor both heteroduplex products. As might be expected, the heterozygoteswith 3 bp deleted (F508del and I507del) resulted in heteroduplex peaksthat were more destabilized than heteroduplex peaks from a single basechange (F508C). The primary peak from the F508C heterozygote was at ahigher temperature than wild type, reflecting the greater stability ofthe T to G transversion (Gundry C N, et al., Genetic Testing, 1999;3:365-70).

Example 7 Mutation Scanning with Saturation Dyes

The HTR2A single nucleotide polymorphism was studied. The PCR wasperformed with KlenTaq, TaqStart, and Tris as described for the cysticfibrosis locus. A 331 bp fragment of the hydroxytryptamine receptor 2A(HTR2A) gene included the common polymorphism (T102C) within exon 1(Lipsky R H, et al., Clin Chem 2001; 47:635-44). The reaction was cycled40 times between 95° C. with no hold, 62° C. with a 2 s hold, and 74° C.with a 20 s hold. A high-resolution melting curve was obtained.

FIG. 6 demonstrates that the saturation dye S5 can be used to scan forsequence variants. That is, the location of the sequence variant neednot be known. The presence of any variant can be detected within a largeamplicon. As seen in FIG. 6, all three genotypes of the singlenucleotide polymorphism in the HTR2A gene (homozygous T, homozygous Cand heterozygous T/C) can be differentiated within a 331 bp amplicon.Melting curve precision and the ability to distinguish differentgenotypes depends on the temperature and fluorescence resolution of theinstrument.

Example 8 Melting Curve Analysis of a DNA Size Ladder: Comparison ofSYBR® Green I to Dye S5

One hundred ng of a DNA size ladder (Low Mass DNA Ladder, Gibco BRL)having six distinct dsDNA species was mixed with either SYBR® Green I(1:10,000) or dye S5 (10 μM) in 3 mM MgCl₂, 100 mM 2-amino-2-methyl-1,3-propanediol, pH 8.7 buffer. A melting curve was obtained on thehigh-resolution instrument at 0.1° C./s.

As discussed above, dye S5, unlike SYBR® Green I, can identifyheteroduplexes in melting curve transitions at concentrations compatiblewith PCR. One reason why SYBR® Green I cannot easily identify lowmelting transitions is illustrated in FIG. 7. When several DNA fragmentsof increasing stability are present, the low temperature peaks are verysmall with SYBR® Green I as compared to dye S5. One explanation is thatduring melting, SYBR® Green I may be released from low temperatureduplexes, only to attach to duplexes that melt at higher temperatures.This causes each successive peak to be higher than the last, with thelowest temperature peaks being very small, if observable at all. Dye S5,which is present at a much higher saturation level, has visible peaksfor even low temperature duplexes. While dye S5 was present at nearsaturation levels in this example, surprisingly, S5 can detect the lowtemperature peaks when diluted to saturation levels of 5-20%. Forexample, the data illustrated in FIG. 13 were obtained using an S5concentration of 1 μM. Thus, while the mechanism is not understood, dyeS5 and various other saturating dyes of this invention do not appear toredistribute during melting.

If the areas of each peak in FIG. 7 are determined and divided by theknown amount of each of the DNA species, the relative sensitivity foreach DNA species can be assessed (FIG. 8). As shown in FIG. 8, with dyeS5, low temperature melting peaks are favored, whereas with SYBR® GreenI, a large enhancement of signal is observed at high temperature.

Example 9 Titration Curves of Common dsDNA Dyes and Determination ofUseful Concentration Range of Dye S5 in PCR

One hundred ng of the low mass DNA ladder was mixed with differentconcentrations of common dsDNA dyes in the presence of 3 mM MgCl₂, 50 mMTris, pH 8.3, 250 μg/ml BSA and 200 μM each dNTP in a final volume of 10al. The samples were transferred to LightCycler® tubes and thefluorescence measured at 40° C. on the real-time fluorimeter. Thefluorescence was normalized to the maximum fluorescence obtained witheach particular dye.

Dilution studies were done using a 152 bp HTR2A amplicon in 10 μlvolumes with 3 mM Mg²⁺, 50 mM Tris-HCl, pH=8.3, 500 μg/ml BSA, 200 μMeach dNTP, 0.5 μM of each primer, 50 ng genomic DNA, 0.4 U of TaqPolymerase, and 88 ng of TaqStart antibody, with S5 dilutions rangingfrom 2 μM to 100 μM. After an initial denaturation for 10 s at 95° C.,40 cycles of 95° C. for 0 sec, 62° C. for 2 sec, and 72° C. for 20 secwere performed. After additional temperature conditioning on theLightCycler® (95° C. for 0 s, 55° C. for 0 s) the samples were melted onthe high-resolution instrument with a slope of 0.3° C./sec.

FIGS. 9A-B show the concentrations of SYBR® Green I and dye S5 that arecompatible with PCR. At concentrations compatible with PCR, SYBR® GreenI is far from saturating the amount of DNA typically present at the endof PCR. Dye S5, in contrast, can be used over a wide range ofconcentrations, including those that are saturating. Typical meltingcurves over a 50-fold range of dye S5 concentration are shown in FIG.10.

Example 10 Fluorescence Spectra of SYBR® Green I and Dye S5

The excitation and emission spectra of SYBR® Green I and dye S5 bound toDNA were measured on a Photon Technology fluorimeter (FL-1). Dye S5 (10μM) or SYBR® Green I (1:10,000) was added to 100 ng DNA (Low Mass DNALadder) in the presence of 3 mM MgCl₂, 50 mM Tris, pH 8.3, 250 μg/ml BSAand 200 μM each dNTP in a final volume of 60 μl.

LightCycler® optics are well matched to SYBR® Green I excitation andemission (FIG. 11). Even though dye S5 is poorly matched to LightCycler®optics, the fluorescence signal observed on the LightCycler® with dye S5at some PCR-compatible concentrations is greater than that usuallyobserved from SYBR® Green I (data not shown).

Many of the other saturation dyes discussed herein are also “blue” dyes.While the fluorescence from such dyes may be observed with standardLightCycler® optics, in some examples, the optics of certain instrumentshave been modified to match the blue dyes better. Such modifications tothe optics are noted in the relevant examples.

Example 11 Genotyping of Beta-Globin Gene Using X-Axis Adjustment andFluorescence Difference Analysis

A 110 bp fragment was amplified from the human beta globin region onchromosome 11 (accession# NG_000007). The 110 bp product included thesites of the common beta-globin mutations HbS and HbC. DNA was extractedfrom dried blood spots of 4 different individuals of each commongenotype. The genotypes included 3 homozygous (AA, SS, and CC) and 3heterozygous (AS, AC, and SC) types. The forward and reverse primerswere ACACAACTGTGTTCACTAGC (SEQ ID NO. 3) and CAACTTCATCCACGTTCACC (SEQID NO. 4), respectively. Each 10 μl reaction contained 50 μg of genomicDNA, 0.50 μM each primer, 10 μM dye S5, 3 mM MgCl₂, 50 mM Tris, pH 8.3,500 μg/ml bovine serum albumin, 0.2 mM each dNTPs, 0.04 U/al Klentaq™(AB Peptides, St. Louis, Mo.), 88 ng TaqStart™ antibody (CloneTech, PaloAlto, Calif.). PCR reaction conditions were as follows: one pre-cyclingdenaturation at 95° C. for 5 sec; 35 cycles of 94° C. for 0 sec, 50° C.for 2 sec, 72° C. for 2 sec with a slope of 2° C. per second. Singlefluorescence acquisitions were taken for each sample after the 2 secextension. After PCR amplification, the samples were cooled at aprogrammed rate of −20° C./sec. Immediately following the rapid cooling,melting was performed on a custom 24-bit high resolution meltinginstrument from 70° C. to 93° C. at a rate of 0.30° C./sec whilecontinuously acquiring fluorescence.

High resolution melting curve data are obtained by measuringfluorescence as the temperature of the sample is increased. The originaldata from quadruplicate samples of 6 genotypes of beta-globin are shownin FIG. 12A. Note that the magnitude of the fluorescence is variablebetween different samples because of sample volume differences andvariable capillary optics.

Magnitude differences between samples can be normalized by using linearbaselines of each curve before and after the major transition.Specifically, two linear regions are selected, one before and one afterthe major transition. These regions define two lines for each curve, anupper 100% fluorescence line and a lower, 0% fluorescence line. Thepercent fluorescence within the transition (between the two regions) iscalculated at each temperature as the percent distance between theextrapolated upper and lower lines. The normalized result for the betaglobin data is shown in FIG. 12B. The quadruplicate samples of eachgenotype clearly group together, most clearly seen in this case around84-85° C. There is still some variation within each genotype, secondaryto temperature offsets between runs (note that there is about a 0.2° C.spread of quadruplicates within genotypes around 10-20% fluorescence).This sample variation can occur between two different samples or evenbetween two different runs of the same sample. Different preparations,including preparations with different salt concentrations, can alsoprovide a temperature offset. However, to at least a firstapproximation, these differences do not affect the shape of the curve.

Temperature offsets between runs can be corrected by shifting thetemperature axis of each curve so that they are superimposed over agiven fluorescence interval. Illustratively, one sample is chosen as astandard, and the points within the florescence interval are fit to aquadratic. For each remaining curve, the required temperature shift fortranslation of each point within the fluorescence interval onto thequadratic is calculated. Each curve is then translated by the averageshift to allow superimposition of the curves within the selectedfluorescence interval. Amplification of a heterozygote produceslow-temperature melting transitions of heteroduplexes as well as highermelting transitions of homoduplexes. If the curves are shifted tosuperimpose their high temperature, homoduplex region (low percentfluorescence), heteroduplexes may be identified by their early drop influorescence at lower temperatures, as seen in FIG. 12C. However, sincethe shape of different homoduplexes does not vary much, temperatureshifting different homoduplexes may obscure any difference between them.

Finally, different genotypes are most easily observed by plotting thefluorescence difference between normalized (and optionally temperatureshifted) melting curves. A standard genotype is first selected(illustratively, the beta-globin wild type AA is used). Then, thedifference between each curve and the standard is plotted againsttemperature, as shown in FIG. 12D. The standard (subtracted from itself)is zero across all temperatures. Other genotypes trace unique paths andcan be identified by visual pattern matching. Automated methods offeature extraction may also be used to assign genotypes. Additionally,while illustrative examples use saturating dyes and heteroduplexdetection, it is understood that temperature shifting and temperaturedifference plots can be used for genotyping when heteroduplexes are notpresent, illustratively for use in viral genotyping wherein the genomeis haploid. Examples of such high resolution genotyping includehepatitis C genotyping, human papilloma virus genotyping, HIVgenotyping, and bacterial identification by ribosomal DNA amplification.

Single parameters that correlate with genotype can be devised. Forexample, normalized curves can be used to determine the temperature atwhich different genotypes are, say 10% melted (90% fluorescence). Thisclearly distinguishes some genotypes, but not others (FIG. 12B).Alternately, the maximum slope of the curve could be used to distinguishhomozygotes from heterozygotes, but different homozygotes are oftensimilar in maximum slope. Finally, the area under the difference curves(FIG. 12D) could be used to define genotype, but such curves can havesimilar area yet trace different paths. While a combination ofparameters may prove to be effective for automated genotypingdetermination, this technique is well suited for visual patternmatching.

It is understood that other normalization techniques are available andare within the scope of the present invention. For example, the HR-1(Idaho Technology, Salt Lake City, Utah) has a setting that willautomatically adjust the fluorescence value at a predeterminedtemperature (illustratively a fluorescence value of 100 at 40° C.), andmelting curves from all samples will be aligned from the samefluorescence value. The difference between the normalization describedabove and this machine-controlled normalization is that with themachine-controlled normalization, the slopes of the curve before andafter the transition are not flattened.

Example 12 Analysis of Larger Amplicons

While short amplicons often result in greater genotyping differences,the dyes of the present invention also may be used to genotype largeramplicons. DNA melting domains are usually about 50 to 500 bp in length,and larger amplicons, for example 500-800 bp, have multiple meltingdomains. A sequence alteration in one domain may not affect melting ofthe other domains, and the variation observed within a domain may beindependent of amplicon length. Thus, while examples are provided in the400-650 bp range, there may be no upper limit to the size of PCR productthat can be scanned for the presence of sequence alterations.

Moreover, because the melting of one domain appears to be independent ofthe melting of other domains, an invariant domain may be used as aninternal control to adjust the X-axis (temperature axis), due toinstrument and/or sample run variation. Heterozygotes aredistinguishable from each other and from homozygotes because the shapesof the melting curves are different. The shapes of the melting curvesare defined by the stability and/or kinetic melting rates of theheteroduplexes and homoduplexes present. Because multiple meltingdomains are present in larger amplicons, the variation in shape mayoccur in any portion of the curve. By adjusting the X-axis positioningof multiple curves to overlap the invariant portion of the curve, thevariable portion of the curve is much easier to discern. Alternatively,by overlapping the variable portion of the curves, if various genotypesare present, the rest of the curves will vary. X-axis adjustmentalternatively could be performed by adding (1) an external controlnucleic acid, or (2) a dye with a second emission wavelength that doesnot interact with nucleic acid but whose fluorescence is dependent ontemperature (a dye with a good temperature coefficient such as Cy5) toeach sample prior to PCR or to melting. Temperature-axis shifting shouldthen be performed according to the position of the melting transition ofthe control nucleic acid or to the intensity profile of the control dye.

FIGS. 13A and 14 illustrate two examples of analysis of largeramplicons. FIG. 13A shows amplification of a 544 bp fragment from thehuman 5-Hydroxytryptamine receptor 2A (HTR2A) gene, exon 2 (accession#NM_000621.1). The forward and reverse primers were CCAGCTCCGGGAGA (SEQID NO. 5) and CATACAGGATGGTTAACATGG (SEQ ID NO. 6), respectively. Each10 μl reaction contained 50 ng of genomic DNA, 0.50 μM each primer, 1 μMdye S5, 2 mM MgCl₂, 50 mM Tris, pH 8.3, 500 μg/ml bovine serum albumin,0.2 mM each dNTPs, 0.4 U Klentaq™ (AB Peptides, St. Louis, Mo.), and 88ng TaqStart™ antibody (CloneTech, Palo Alto, Calif.).

PCR reaction conditions were as follows: 40 cycles of 92° C. for 0 sec,60° C. for 2 sec, 74° C. for 25 sec. After PCR amplification, thesamples were cooled at a programmed rate of −20° C./sec. Immediatelyfollowing the rapid cooling, melting was performed on a custom 24-bithigh resolution melting instrument from 70° C. to 93° C. at a rate of0.30° C./sec while continuously acquiring fluorescence.

Duplicate samples of each genotype (CC, TC, and TT) were amplified andanalyzed, as shown in FIG. 13A. The data were normalized and temperatureshifted as described in Example 11, except that curves were superimposedbetween 10 and 20% fluorescence. FIG. 13B shows a predicted melting mapof the homoduplex and the position of the polymorphism in the lowermelting domain. The experimental data show two apparent melting domains.All genotypes are similar in the higher melting domain. Genotypes differin the lower melting domain, where the heterozygote shows typicalbehavior of low melting heteroduplexes with the heterozygote curvecrossing the lower melting homozygote curve and approximation to thehigher temperature homozygote with increasing temperature.

FIG. 14 shows difference curves for amplification of a 612 bp fragmentfrom the cystic fibrosis transmembrane conductance regulator (CFTR)gene, exon 10 (accession#M55115). The forward and reverse primers wereAGAATATACACTTCTGCTTAG (SEQ ID NO. 7) and TATCACTATATGCATGC (SEQ ID NO.8), respectively. Each 10 μl reaction contained 50 ng of genomic DNA,0.50 μM each primer, 10 μM dye S5, 3 mM MgCl₂, 50 mM Tris, pH 8.3, 500μg/ml bovine serum albumin, 0.2 mM each dNTPs, 0.4 U Klentaq™ (ABPeptides, St. Louis, Mo.), and 88 ng TaqStart™ antibody (CloneTech, PaloAlto, Calif.). PCR reaction conditions were as follows; 35 cycles of 89°C. for 0 sec, 58° C. for 8 sec, 74° C. for 35 sec. Single fluorescenceacquisitions were taken for each sample after the 35 sec extension.After PCR amplification, the samples were cooled at a programmed rate of−20° C./sec. Immediately following the rapid cooling, melting wasperformed on a custom 24-bit high resolution melting instrument from 60°C. to 87° C. at a rate of 0.30° C./sec while continuously acquiringfluorescence. In this example, heterozygote differentiation was bestwhen the middle part of the curve (30-40% fluorescence) is used forX-axis adjustment. Finally, the fluorescence of each plot was subtractedfrom one of the wild type plots to give the difference plots shown inFIG. 14. Each sequence alteration is clearly different from the wildtype and all genotypes can be differentiated.

Example 13 Targeted Detection and Multiplexing with Saturation Dyes

The dyes of the present invention may be used as a donor to excite anacceptor dye attached to an oligonucleotide probe. Because these dyesmay be used at or near saturating concentrations to bind to thehybridized probe at a high density (approximately two dye moleculesevery three base pairs), the dye is available throughout the length ofdouble-stranded DNA for fluorescence resonance energy transfer. A probewith an acceptor dye is added to the reaction before PCR, amplified andis detected when hybridized to the product. The binding of thesaturation dye at high density to the duplex provides favorableexcitation to the acceptor dye on the probe, producing a high degree ofacceptor fluorescence. Previously, dyes with a high bp/dye ratio wereused and only produced low levels of acceptor fluorescence.

Multicolor experiments can be performed with multiple probes. Forexample, total amplicon melting can be monitored at 470 nm, the emissionof a fluorescein-labeled probe could be monitored at 515, a HEX-labeledprobe (that hybridizes to a different segment of DNA internal to theprimers) monitored at a third wavelength, and a TET-labeled probe (thathybridizes to yet a different segment internal to the primers) monitoredat a 4th wavelength. Color compensation, as is well known in the art, isused to deconvolute the overlapping four signals. The result is that thefirst signal can be used to scan for mutations over the whole amplicon,while the 2nd, 3rd, and 4th signals allow genotyping of smaller regionswithin the amplicon.

Example 14 High Resolution Melting Curve Analysis for GenotypeComparison

Dyes of the invention can be used to determine whether any twoindividuals share the same alleles on a gene fragment. In the previousexamples, the genotype (including the exact allele, heterozygosity, andhaplotype) of a reference sample was known. In some applications, theexact genotype of a reference sample need not be known, as long ashigh-resolution melting curve analysis makes it possible to determinewhether a sample of another individual (or of unknown origin) is thesame as the reference. An illustrative example is the identification ofHLA alleles shared among family members.

Human Leukocyte Antigens (HLA) are cell surface proteins of white bloodcells and other tissues of the body which play a key role in immunerecognition, and thus in transplant tolerance or rejection. Matching ofHLA alleles between donor and recipient is important for organtransplant. HLA proteins form two major groups: class I, and class II.Each group is encoded by multiple genes. The currently acceptedtechniques for determining the HLA allelotype of a tissue includeserotyping with specific antibody reagents, hybridization with nucleicacid probes, and direct sequencing of the HLA genes. Because a largenumber of genes and loci need to be tested, the cost to determine theHLA allelotype is over $1,000 per person. Complete genotyping of HLA isnecessary when donor and recipient are unrelated. However there is abouta 25% chance of a perfect HLA match between siblings and for this reasonorgan transplant between siblings is preferred when HLA matches indicatethat it is possible. In this case it is only necessary to demonstratethat the donor and recipient relatives share the same HLA alleles.Determining the exact identity of the shared alleles is not necessary.

Genomic DNA samples of CEPH/Pedigree Utah family 1331 were obtained fromthe Coriell Institute. There are 17 people across three generations inthis family including four internal grandparents, two parents, andeleven children (pedigree of family 1331 is shown in FIG. 15). Two othersamples with well known homozygous genotypes of HLA-A BM15(0101) andBM16(0202) were also obtained from Coriell.

Amplification of two exons of the HLA-A gene were performed as follows:HLA class I genes are so similar over of the length of their codingexons that it is difficult to design PCR primers that amplify only theHLA-A gene and not the related class I genes. A nested PCR strategy wasadopted in which an initial round of PCR specifically amplified a large(948 bp) fragment of the HLA-A gene followed by secondary amplificationof that product using internal primers. The primers used in the firstPCR hybridized to HLA-A intron 1 (forward primer5′-GAAAC(C/G)GCCTCTG(C/T)GGGGAGAAGCAA (SEQ ID NO. 9, SEQ ID NO. 10, SEQID NO. 11, SEQ ID NO. 12)) and intron 4 (reverse primer5′-TGTTGGTCCCAATTGTCTCCCCTC (SEQ ID NO. 13)). In the secondary PCRs theforward primers 5′AGCCGCGCC(G/T)GGAAGAGGGTCG (SEQ ID NO. 14, SEQ ID NO.15) and reverse primer 5′GGCCGGGGTCACTCACCG (SEQ ID NO. 16) were used toamplify a 335 bp segment of HLA-A exon 2. The forward5′CCC(G/A)GGTTGGTCGGGGC (SEQ ID NO. 17, SEQ ID NO. 18) and reverseprimer 5′ATCAG(G/T)GAGGCGCCCCGTG (SEQ ID NO. 19, SEQ ID NO. 20) wereused to amplify a 366 bp fragment of HLA-A exon 3. In the primersequences of this example, (N/N′) represents that the primer is amixture of nucleotide sequences having equal percentages of N and N′ atthat position. For example, the forward primer for the 335 bp segment ofHLA-A exon 2 contains an equal mixture of two nucleotides, with either aG or an A at the fourth position, as represented by SEQ ID NO. 17 andSEQ ID NO. 18. The forward primer for the HLA-A intron 1 has two suchsites, and thus is an equal mixture of four nucleotides, as representedby SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12.

All PCRs were performed in glass capillaries using the LightCycler®. Theinitial PCR contained 0.5 μM forward and reverse primers, 50 ng genomicDNA in a buffer of 3 mM Mg^(++,) 50 mM Tris-HCl pH 8.3, 500 μg/ml BSAand 20 μM of dye D6 in 10 μl. Cycling conditions were 94° C. for 20 sfollowed by 40 cycles of 94° C. 1 s, 62° C. for 0 s, 72° C. for 1 min.The secondary, nested PCRs contained 0.25 μM forward and reverse primer,1/10000 of first PCR product in the same buffer containing 2 mM Mg⁺⁺.Cycling conditions were 94° C. for 5 s followed by 25 cycles with 94° C.1 s, 65° C. for 0 s, 72° C. for 8 s.

After the secondary amplification the glass capillaries were transferredto the high resolution melting instrument HR-1, and a melt wasperformed. The sample was heated from 60° C. to 95° C. at a rate of 0.3°C./s and fluorescence (450 excitation/470 emission) and temperaturemeasurements were acquired every 40 s (FIGS. 16A-B). The nestedamplification products were sequenced by the ABI 3700. Sequencherversion 4.0 was used for the sequence analysis.

Concordance of melting curve analysis and sequencing results weredetermined as follows: Melting curve analysis of the exon 2 and exon 3PCR products amplified from the 17 members of the CEPH/Pedigree Utahfamily 1331 clustered in six different groups (FIGS. 16A-B). Thissuggested that there are six different HLA-A genotypes in this family.The exon 2 and exon 3 PCR products were sequenced, and the resultsconfirmed the melting curve analysis, identifying the six genotypes as:HLA-A 02011/3101 (herein referred to as genotype AB) for family members1, 4, 7, 12; HLA-A 3101/2402101 (genotype BC) for family members 3, 5,6, 11, 17; HLA-A 02011/2402101 (genotype AC) for family members 2, 9,10, 16, HLA-A 02011/03011 (genotype AD) for family members 13, 14; HLA-A02011/02011 (genotype AA) for family member 8 and HLA-A 2402101/01011(genotype CE) for family member 15 (Results for exon 2 is shown in FIGS.16A-B).

In some cases, the amplification products from siblings may showidentical or nearly identical melting curves despite having differentgenotypes. In such cases mixing the genomic DNA from the two siblingsbefore the initial PCR followed by the two amplification steps andmelting curve analysis can differentiate identical from non-identicalgenotypes. In particular if the siblings have identical genotypes, themixed melting curve will be identical to those performed separately. Ifsiblings have different genotypes then the mixed melting curve will bedifferent from that of the individual melting curves. Mixing experimentswithin each group confirmed that the members of each group sharedidentical genotypes.

Another example of the mixing analysis technique was demonstrated by twohomozygous samples BM15 (0101) and BM16 (0201). In this case, the twoalleles have a total of 15 nucleotide differences spread over the lengthHLA-A exon 2, but they show similar melting curves. The melting curve ofthe mixed samples was significantly shifted to the left (lower meltingtemperature) due to the 15 mismatches present in the heterohybridsgenerated in the mixed sample PCR from HLA-A exon 2 (see FIG. 17).

Example 15 Monitoring Amplification in Real-Time with Saturating Dyes

A 60 bp fragment of the HTR2A gene was amplified with forward andreverse primers ACCAGGCTCTACAGTAA (SEQ ID NO. 21) and GTTAAATGCATCAGAAG(SEQ ID NO. 22), respectively. Amplification was performed using thereagents described in Example 12 but with modifications to the cyclingparameters, which were 95° C., 0 s; 62° C., 2 s; 74° C., 20 s using theLightCycler®. Various concentrations of SYBR® Green I, D6, Z6, and N7were independently present in the reaction mixture. Fluorescence datawere acquired once each amplification cycle, up to 36 cycles.Fluorescence crossing points (Cp), calculated as the second derivativemaximum of the amplification plot (cycle number plotted on the x-axisagainst fluorescence intensity on the y-axis), were obtained as follows:

TABLE 4 Dye present in reaction Dilution/Concentration Cp SYBR ® Green I1:2,500  No amplification 1:5,000  26 1:10,000 26 1:20,000 Signal tooweak D6 250 μM  No amplification 125 μM  28  63 μM 27  31 μM 27  16 μM26   8 μM 26   4 μM 26   2 μM 26   1 μM Signal too weak Z6  20 μM Noamplification  10 μM 31   5 μM 27 2.5 μM 26 1.3 μM 26 0.7 μM 25 0.4 μM25 0.2 μM Signal too weak N7  62 μM No amplification  31 μM 27  16 μM 277.9 μM 26 4.0 μM 26 2.0 μM 26 1.0 μM 26 0.5 μM 26 0.2 μM 26 0.1 μMSignal too weakThe Cp value, which represents the cycle number at which signal risesabove background, is expected to increase when inhibitors present in thereaction affect the efficiency of amplification. Under the conditions ofthese experiments, however, inhibition by increasing amounts of dyeresulted not as a gradual increase in Cp, but as a sudden and completeelimination of amplification. Due to the small size of the amplicon(which results in a lower signal compared to larger amplicons), SYBR®Green I dye could only be used in the range of two-fold concentrationsfor real-time monitoring. In contrast, dyes D6, Z6 and N7 could be usedin the range of 32 to 128-fold concentrations. It is contemplated thatmany saturating dyes have a wide range of concentration that can be usedin real-time monitoring of amplification.

Example 16 SNP Typing by Use of Unlabeled Probes and Saturating Dyes

As shown in FIG. 7, saturation dyes have the ability to detect meltingsignatures of multiple dsDNA species present in a reaction mixturewithout having low temperature melting obscured by redistribution of dyefrom low to high melting temperature (Tm) duplexes. This aspect ofsaturation dyes allows the use of unlabeled probes for genotyping. Whenan unlabeled probe is mixed with the amplicon in the presence of asaturation dye, the melting signature of both the amplicon and theprobe-target duplexes can be observed at the same time. Changes inmelting profile can be used to detect the presence of sequence variancesunder the probe, as well as elsewhere in the amplicon. Optionally, bytruncating the melting process prior to the melting transition of theamplicon, one can study just the melting of the unlabeled probe. The useof unlabeled probes for effective genotyping and mutation scanning hasnot been possible with dyes that are currently used for real-time PCR,such as SYBR Green I (compare FIG. 21B versus 21C). Further, because ofthe properties of the saturation dyes, genotyping can be performed inthe presence of the unlabeled probe without need for the unlabeled probeor the target nucleic acid to be immobilized on a surface. In each ofthe illustrative embodiments, the dye, unlabeled probe, and targetnucleic acid are all free in solution.

A 300 bp amplicon was generated by PCR in a LightCycler® (Roche AppliedSystems, Indianapolis, Ind.) using primers 5′GATATTTGAAGTCTTTCGGG (the“reverse” primer, SEQ ID NO. 23) and 5′TAAGAGCAACACTATCATAA (the “sense”primer, SEQ ID NO. 24) in a 10 μl reaction mixture that contained aninitial template plasmid DNA (Example 5) of 1×10⁶ copies, 0.5 μM 3′-endphosphorylated probe, 3 mM MgCl₂, 50 mM Tris, pH 8.3, 0.2 mM each dNTP,500 μg/ml BSA, 20 μM dsDNA dye D6 (Example 1) and 0.4 U Taq polymerase(Roche). For symmetric amplification, 0.5 μM of each primer was used.For asymmetric amplification, primer ratios were varied between 10:1,20:1 and 50:1. PCR was performed with an initial denaturation at 95° C.for 10 s, followed by 45 cycles of 95° C. for 1 s, 55° C. for 0 s, 72°C. for 10 s (or 5 s in the absence of probe). At the end ofamplification, samples were denatured at 95° C. for 0 s, annealed at 40°C. for 0 s followed by melting analysis at a rate of 0.2° C./s up to 90°C. (LightCycler®), or alternatively, up to 75° C. at a rate of 0.3° C./s(HR-1) or 0.1° C./s (LightTyper® with modified optics of 450 nmexcitation and 470 nm detection).

The target strand of the amplified product should be adequatelyavailable for probe hybridization. In many instances this may beaccomplished by providing the primer that generates theprobe-hybridizing strand (the “sense” primer) in excess compared to theother primer (the “reverse” primer). FIGS. 18A and 19 show results of anoptimization experiment in which amplicons generated by varying ratiosof primers were examined. While the optimum primer ratio may bedifferent for each amplicon and/or each amplification system, melting ofthe probe is often observed when the ratio between sense and reverseprimers is about 10 to 1 or higher. Note that in the LightCycler® data,melting peaks of both the probe and the amplicon are observed at such aprimer ratio (FIG. 18B). It is understood that, while the examplespresented herein use this “asymmetrical” PCR to generate a greaterabundance of the target strand, other methods of amplification may bewell suited, particularly amplification methods such as NASBA, TMA, androlling circle amplification that favor amplification of one strand.Alternatively, strand separation may occur subsequent to PCR,illustratively by incorporating a biotin tail or a poly-A tail (or othersequence) in the amplicon through use of properly designed primers. Inyet another alternative example, melting analysis may be conducted onsingle-stranded nucleic acid subsequent to or in the absence ofamplification. Also, it has been found that the relative magnitude ofthe probe-target transition increases as the amplicon length decreases.Accordingly, in still another example shorter amplicons may be used,illustratively 100 bp or shorter.

In the present examples, the 3′ end of the unlabeled probe isphosphorylated to prevent polymerase extension during amplification. Ifdesired, polymerase extension of the probe may be prevented by othermeans, including using a 2′,3′-dideoxynucleotide, a 3′-deoxynucleotide,a 3′-3′ linkage, other non-extendable termination such as the 3′-spacerC3 (Glen Research, Sterling, Va.), a biotin with an optional linker, ora minor-groove binder. Mismatching of two or more of the 3′ terminalbases of the probe can also be used to prevent extension. Alternatively,the unlabeled probe with or without a blocked 3′-end could be added tonucleic acid sample mixtures that substantially lack polymeraseactivity.

Another consideration for successful genotyping is the length and GCcontent of the probe. As the binding mode of saturation dyes to dsDNA isnot yet completely understood, several probe designs are tested. Table 5and FIG. 20 show an optimization experiment using dye D6 in which probesdiffering in length from 14 to 30 bases with GC of 14 to 37% wereexamined. When the probe and target are completely complementary,melting peaks of probes as short as 14 bases were detected (FIG. 20).However, when there was a mismatch under the probe (which in thisexample is positioned in the middle of the probe), melting peaks werenot observed for probes shorter than 22 bases (not shown), suggestingthat dye D6 requires at least 10-11 bp of uninterrupted binding spaceunder these conditions. In the design of probes, mismatches optionallycan be positioned closer to the ends of the probe, as well as in themiddle of the probe. A similar experiment was conducted with probesequences of 100% AT and 100% GC hybridizing against syntheticcomplementary strands. The melting peaks of these probes were clearlydetected when probes were 24 bases or longer (data not shown) with the100% AT probe, and as short at 10 bases with the 100% GC probe. Resultsof melting analysis on the LightCycler®, HR-1, and the modifiedLightTyper® were all in agreement.

TABLE 5 SEQ  Length GC ID (bases) Probe Sequence % NO. 14         caatgaa*tatttat 14.3 SEQ   ID NO.  25 16        tcaatgaa*tatttatg 18.8 SEQ   ID NO.  26 18       ttcaatgaa*tatttatga 16.7 SEQ   ID NO.  27 20      attcaatgaa*tatttatgac 20 SEQ   ID NO.  28 22    g attcaatgaa*tatttatgac g 27.3 SEQ   ID NO.  29 24   gg attcaatgaa*tatttatgac ga 29.2 SEQ   ID NO.  30 26  ggg attcaatgaa*tatttatgac gat 30.8 SEQ   ID NO.  31 28 gggg attcaatgaa*tatttatgac gatt 32.1 SEQ   ID NO.  32 30ggggg attcaatgaa*tatttatgac gattc 36.7 SEQ   ID NO.  33 a* denotesposition complementary to the SNP on the amplicon

SNP typing was demonstrated with 28-base probes and templates that areeither homozygous for A, C, G, and T at the alternative base position orare heterozygous for that position as follows: A/C, A/G, A/T, C/G, C/T,and G/T. FIGS. 21A-D show the melting curves generated by a probe thatis fully complementary to the A homozygote (in this case the probesequence is A and the sense strand is T). All homozygotes melted in asingle transition (FIG. 3A). The A::T match is most stable with a Tm of66.1° C. (predicted 64.0° C.), with the mismatches A::G (63.0° C.,predicted 62.4° C.), A::A (61.9° C., predicted 60.7° C.) and A::C (61.4°C., predicted 60.8° C.) decreasing in order of stability. The Tmpredictions did not account for the presence of the dye. As is the casewith amplicon Tm (FIG. 10), the presence of dye usually increases probeTm. Melting curves for all homozygotes are clearly separated anddistinguishable (FIG. 21A). Heterozygous templates were separated intotwo groups: those in the first group had an allele fully complementarywith the probe (A/T, A/C, A/G), and those in the other group did not(C/T, C/G, G/T). In the first group, the probe melting curve clearlydisplayed two peaks: the higher Tm peak matching with the homozygous Atemplate, and the lower Tm peak characterizing the type of base mismatch(FIG. 21B). In the second group, melting curves showed only one meltingpeak, each of which was shifted to the left compared to the homozygous Atemplate, and each of which is readily distinguishable from the others.(FIG. 21D). When the same test was done with SYBR Green I dye, themelting peaks of homozygous templates having a mismatch with the probeshifted to the left compared to the perfectly matched homozygous Atemplate, but they were indistinguishable from each other. Heterozygoustemplates C/T, C/G, G/T were also shifted to the left, but not separatedfrom each other. The melting curves of heterozygous templates A/C, A/G,and A/T could not be distinguished from the homozygous A template (FIG.21C).

Example 17 Cystic Fibrosis Genotyping with Unlabeled Probes andSaturating Dyes

Fragments of the CFTR gene exon 10 and 11 were amplified using samplesobtained from Coriell Institute for Medical Research, and primers5′ACATAGTTTCTTACCTCTTC (SEQ ID NO. 34, sense primer), and5′ACTTCTAATGATGATTATGGG (SEQ ID NO. 35, reverse primer) for exon 10, andprimers 5′ TGTGCCTTTCAAATTCAGATTG (SEQ ID NO. 36, sense primer) and 5′CAGCAAATGCTTGCTAGACC (SEQ ID NO. 37, reverse primer) for exon 11. Theprobe for exon 10, which hybridizes over the F508del mutation, was5′TAAAGAAAATATCATCTTTGGTGTTTCCTA (SEQ ID NO. 38). Two probes were usedfor the detection of the G542X mutation in exon 11(5′CAATATAGTTCTTNGAGAAGGTGGAATC, SEQ ID NO. 39), where N is either G orT. All probes were phosphorylated at the 3′ end. PCR was performedasymmetrically, using a primer ratio of 10:1 (0.5 μM sense; 0.05 μMreverse). Other reagents for PCR were essentially the same as in Example16, except 50 ng of genomic DNA was used as template. Cycle conditionswere 95° C. for 10 s and then 45 cycles of 95° C. for 0 s, 52° C. for 0s and 72° C. for 10 s. Melting curve analysis on the LightCycler® wasperformed as in Example 16. Table 6 lists the various deletions detectedby the probes.

TABLE 6 Mutation Nucleotide position Amino acid change exon I506V A or Gat 1648 Ile or Val at 506 10 I507 del deletion of 3 bp between deletionof Ile506 or 10 1648 and 1653 Ile507 F508del deletion of 3 bp betweendeletion of Phe at 508 10 1652 and 1655 F508C T or G at 1655 Phe or Cysat 508 10 G542X G to Tat 1756 Gly to Stop at 542 11

The F508del mutation was chosen as an example of a small (3 bp)deletion. The probe contained a 28 base wild type sequence. FIG. 22Ashows that the Tm of the homozygous F508del shifted about 10° C. belowthe wild type. The melting curve of the heterozygous F508del showed twomelting peaks: one at the same temperature as the wild type and theother the same as the homozygous F508del. FIG. 22B shows an overlay ofthree additional heterozygous mutations found in this locus, F508C,I506V, and 1507 del.

The G542X mutation is a single base mutation in which the wild type Gchanges to T. The 28 base wild type probe G and mutation probe T wereused to type G542X homozygous (T/T) and heterozygous (G/T) mutationswithout sequencing (FIG. 23).

Example 18 Genotyping by Multiple Unlabeled Probes and a Saturating Dye

Melting analysis using an unlabeled probe with a saturating dye enablesgenotyping of one or more sequence variants under the probe. The use ofmultiple unlabeled probes enables simultaneous genotyping of sequencevariants in multiple sequence segments. Probes can be designed tohybridize with multiple sequence segments on one DNA fragment, or withsequence segments on multiple DNA fragments. Illustratively, wheremultiple probes are used with a single target DNA fragment, the probesdo not need to overlap to provide information on sequence variation. Inan illustrative example, a 210 bp fragment of the cystic fibrosis genewas asymmetrically amplified in the presence of 20 μM of dye D6 or N7,using two primers described earlier (the sense primer SEQ ID NO. 34 andthe reverse primer SEQ ID NO.35 at 0.5 M and 0.05 μM, respectively). Inthis 210 bp fragment, the presence or absence of two mutations, F508 deland Q493V, were tested by two unlabeled probes: Probe 1:ATCTTTGGTGTTTCCTATGATG (SEQ ID NO. 48; underlined are the three basesthat become deleted in the F508del mutation) and Probe 2:CTCAGTTTTCCTGGATTATGCCTGGC (SEQ ID NO. 49: underlined is the base thatmutates into T in Q493V). The 3′ ends of both probes werephosphorylated. The melting temperatures of the matched or mismatchedprobes were kept below 72° C. to allow sufficient separation from theamplicon melting signature. Melting analysis was conducted with theLightCycler®, HR-1, and modified LightTyper® instruments, as in Example16. The melting data from all three instruments correctly genotyped foursamples: wild type, F508del homozygote, F508del heterozygote, andF508del/Q493V compound heterozygote. FIG. 24 shows the melting profilewith dye N7. Very similar results were obtained with dye D6.

Example 19 Simultaneous Mutation Scanning and Genotyping

Testing for many genetic disorders can be difficult because thecausative mutations are often scattered all over the gene. Simultaneousmutation scanning and genotyping can be used to detect thesealterations. Illustratively, for any particular gene of interest, eachexon is amplified using primers outside of common splice sites. If highfrequency sequence variants are known, an unlabeled probe may beincluded to genotype the site, or an additional set of primers may beuse to amplify this smaller locus. Mutation scanning and genotyping ofspecific sites can be performed in separate reactions, or when scanningis positive, the probe subsequently can be added to the tube and meltinganalysis repeated for genotyping. Preferably, scanning and genotypingare done simultaneously by analyzing both full-length amplicon andsmaller locus duplexes in the same melting curve analysis. Because ofthe different sizes of the full-length amplicon and the smaller locus,it is expected that melting peaks for the full-length amplicon would beat higher temperatures than for the smaller locus.

The average gene covers about 27 kb, but only about 1,300 bases code foramino acids. On average, there are about 9 exons per gene with a meanlength of about 150 bp. Of the sequence alterations that cause disease,about 70% are SNPs, with 49% missense, 11% nonsense, 9% splicing and <1%regulatory mutations. Small insertions/deletions make up 23% ofdisease-causing mutations. The remaining 7% are caused by largeinsertions or deletions, repeats, rearrangements, or compound sequencealterations. Some sequence alterations do not affect gene function, forexample silent SNPs that result in the same amino acid sequence.Additional examples include SNPs and in-frame insertions or deletionsthat change the amino acid sequence but do not alter protein function.Most SNPs and repeats within introns do not cause disease, except forsplicing and regulatory mutations. With the exception of large deletionsand sequence alterations deep within introns, disease-causing mutationscan be identified by PCR using primers within introns that flank eachexon. The primers are placed outside of likely splice site mutations. Ifthe sequence alteration is not amplified, it will not be detected by anymethod, including sequencing.

High-resolution melting analysis using the dyes of the presentdisclosure becomes more difficult as the amplicon size increases.Optionally, to maintain scanning sensitivity near 100%, exons greaterthan 400-500 bases can be scanned with more than one amplicon.Illustratively, common thermal cycling parameters are used so that allexons can be amplified at once.

Simultaneously with mutation scanning, common mutations andpolymorphisms can be genotyped by including one or more unlabeledoligonucleotide probes or by selectively amplifying a smaller ampliconusing one or more additional sets of primers. Amplification of both thelarger and the smaller amplicon can be performed simultaneously, or by abiphasic PCR which amplifies the larger amplicon in the first phase, andthe smaller amplicon(s) in the second phase. This biphasic PCR can beachieved by designing the Tm and amount of each of the primers in a waythat, by adjusting the annealing temperatures of the two phases,preferentially amplification of the different amplicons occur. When thesignal from the larger amplicon is expected to overwhelm or mask thesignal from the shorter amplicon, this biphasic technique can be used toadjust the final amount of each of the amplicons to circumvent such aproblem. When one or more oligonucleotide probes are used, amplificationof the larger amplicon by mild asymmetric PCR can be used,illustratively having about a 10:1 primer ratio.

An illustrative example of using a single melting procedure forsimultaneously scanning for mutations in the amplicon and genotyping bya probe was conducted as follows: The cystic fibrosis exon 11 fragmentwas amplified from ten wild type and twenty unknown specimens in thepresence of the wild type probe G and dye D6 according to the methoddescribed in Example 17. The amplified samples were then melted on themodified LightTyper instrument (Example 16) using a ramp rate of 0.1°C./s between 40° C. and 90° C. FIG. 25A shows the amplicon portion ofthe melting curve (75-83° C.). Ten of the samples had slightlyleft-shifted curves that were clearly distinct from the ten wild typesamples. These were suspected of carrying a heterozygous mutationsomewhere in the amplicon. The rest of the curves followed the predictedshape of homozygous samples. However, the curves of the remaining tenunknown samples did not overlap with the wild type curves, andtherefore, the majority of these were flagged as possible sequencevariants as well. FIG. 25B shows the probe portion of the melting curve(58-72° C.) plotted as the negative derivative. Here, each of the thirtysamples were unambiguously genotyped.

Example 20 Monitoring Fluorescence During Amplification Using UnlabeledProbes

When fluorescence from a dsDNA-binding dye is monitored during PCR, itis possible to observe, cycle by cycle, the generation of a specifictarget nucleic acid sequence as defined by hybridization of the targetnucleic acid to an unlabeled probe. The amount of target sequence ateach PCR cycle depends on its initial amount in the original sample.Therefore, quantification is possible. As illustrated, the fluorescencesignals from the amplicon and other dsDNA are separated from theprobe-specific signal. This is achieved by monitoring fluorescence at aplurality of temperature points comprising before and within the probemelting transition, during at least two cycles in an amplificationreaction.

In an illustrative example, a 300 bp fragment of the DNA Toolbox plasmid(Example 5) was amplified in the presence of a 28-bp probe (Table 5, SEQID NO. 32) using reagents described in Example 16, except that thetemplate DNA was 10⁵ copies/10 μl, unless otherwise stated, MgCl₂ wasused at 2 mM, and dye N7 was used instead of D6. A primer ratio of 10:1was used. The probe and the plasmid were matched in sequence (i.e. nomismatch under the probe). Amplification was performed in theLightCycler using the following programmed parameters for 45 cycles: 95°C. for 0 s, cooling at −20° C./s, 52° C. for 0 s, heating at 0.5° C./s,74° C. for 8 s, heating at 20° C./s. Fluorescence was continuouslymonitored between 52° C. and 74° C. as shown in FIG. 26 during eachcycle of PCR. The data files were then imported into a custom software,essentially as described in U.S. Pat. No. 6,174,670, herein incorporatedby reference, to analyze the multiple fluorescence data obtained eachcycle. The quality of amplification was assessed by the traditionalmethod of plotting the fluorescence value at one temperature each cycle,such as 61° C. at which the amount of all dsDNA can be observed (FIG.28A closed square), or 73° C. at which only the amount ofdouble-stranded amplicon is observed (FIG. 28A open square).

When a temperature range specific for observing probe melting wasselected for each cycle (in this case 62-72° C.), the resultingfluorescence curves (plotted as the negative derivative in FIG. 27)showed that with increasing cycle number, the melting peak of the probegrew larger. One method to express the correlation between cycle numberand the amount of probe:target specific signal is to plot the differencein derivative value at the probe Tm (in this case at 66.5° C.) and justbefore the probe's melting transition (at 64° C.). The resulting plot isshown in FIG. 28B (open triangle) showing a positive correlation betweenprobe:target signal and cycle number. The two temperature points can bepredetermined, or chosen visually from the derivative curves, orobtained from the second derivative curves where the second derivativeequals zero. It is contemplated that, once such plots are established,these plots can be used for quantification of initial template in asample.

In another method, the curves in FIG. 27 were baseline subtracted andfit to a Gaussian (when possible) so that the area under the peaks canbe calculated and plotted against cycle number as in FIG. 28B (closedtriangle). In FIG. 28C this was further shown with samples of threedifferent initial template concentrations, each fitted to a straightline indicating a positive and linear correlation between cycle numberand specific amplification product as defined by the probe. Melting peakarea values were negative during the few cycles prior to when theystarted to increase with cycle number. This provided the opportunity toset crossing points at zero peak area, although even if the peak areavalues did not cross zero, extrapolation to zero can be used. When thesecrossing points were plotted against the log of the initial templateconcentration, a linear relationship was found (FIG. 28D) indicatingthat it is possible to use melting of the unlabeled probe forquantification of the initial template by fluorescence monitoring duringamplification.

While this and other examples use saturating dyes, it is understood thatother dyes may be used with certain methods described herein,particularly with methods where detection of SNPs and other smallchanges is not necessary. For example, in the above method, the probematched perfectly to the target sequence, and the method did not includedetection of genetic variation. However, it is also understood that theabove method can be used in combination with various other analyses,including genotyping.

Example 21 Detection of Mutations in the c-Kit Gene for the Diagnosis ofGIST

The human c-kit protein is a transmembrane receptor tyrosine kinasewhich is activated through binding of its ligand. Activation of thetyrosine kinase leads to autophosphorylation of tyrosine residues which,in turn, leads to an intracellular signaling cascade resulting in cellproliferation. Mutations that cause activation of the c-kit proteinindependent of ligand binding have been observed in a variety of tumorsincluding germ cell tumors, mast cell tumors and stromal tumors of thegastrointestinal tract (GISTs). These mutations are thought to be thedriving force for neoplastic growth. One recent success in targetedmolecular therapy is the development of the drug STI-571 (imatinib,gleevec) which inhibits the activated c-kit receptor in GISTs. This drugis a phenylaminopyrimidine derivative, and many GIST patients treatedwith STI-571 show partial responses and stabilization of disease.However, there is a need to provide an improved diagnosis for this classof neoplasm.

Historically, the diagnosis of stromal tumors in the gastrointestinaltract has been difficult. Currently, stromal tumors of thegastrointestinal tract are routinely immunostained for CD117 (c-kit). Apositive immunostain suggests that the tumor is correctly identified asa GIST. However, sometimes the immunostain for c-kit is focal and/orequivocal and difficult to evaluate. Furthermore, the immunostain doesnot give information on the location, presence or absence, and type ofan activating mutation, and commercially available CD-117 antibodies maycross-react with non c-kit molecules. High-resolution amplicon meltingcan be used as an improved method to rapidly screen for activatingmutations in the c-kit gene. Sequence variants detected byhigh-resolution melting can optionally be further characterized by DNAsequencing.

An illustrative experiment for c-kit screening was performed as follows:A variety of GIST tissue specimens, including primary tumor,metastatic/recurrent tumor, neoplasms arising from the small and largeintestine, stomach, peritoneum, and per-pancreatic soft tissue wereobtained in paraffin blocks. DNA was isolated from sectionedparaffin-embedded tissue on glass slides by first de-paraffinizing andrehydrating the samples by successive washes in xylene, and then in 95%,70%, 50% and 30% ethanol. After final rinsing in deionized H₂O, theslide was dried under an infra red lamp for 5 min. The appropriate areaof tumor tissue was microdissected off the slide with a scalpel andincubated in 50 to 100 μl of 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1%Tween 20, 1.0 mg/ml proteinase K overnight at 37° C. The sample was thenincubated in a boiling H₂O bath for 10 min to inactivate the proteinaseK. After cooling on ice, the sample was diluted in 10 mM Tris-HCl (pH7.5), 0.1 mM EDTA and subjected to polymerase chain reaction (PCR) withthe exon-specific primers shown in Table 7.

TABLE 7 Primer Sequence (5′ → 3′) SEQ. ID. NO. Exon 9 ForwardGATGCTCTGCTTCTGTACTG SEQ ID NO. 40 Exon 9 ReverseGCCTAAACATCCCCTTAAATTGG SEQ ID NO. 41 Exon 11 ForwardCTCTCCAGAGTGCTCTAATGAC SEQ ID NO. 42 Exon 11 ReverseAGCCCCTGTTTCATACTGACC SEQ ID NO. 43 Exon 13 ForwardCGGCCATGACTGTCGCTGTAA SEQ ID NO. 44 Exon 13 ReverseCTCCAATGGTGCAGGCTCCAA SEQ ID NO. 45 Exon 17 Forward TCTCCTCCAACCTAATAGTGSEQ ID NO. 46 Exon 17 Reverse GGACTGTCAAGCAGAGAAT SEQ ID NO. 47

PCR was performed in a total volume of 20 μl in a capillary cuvette. Thereaction mixture contained 50 mM Tris-HCl (pH 8.5), 3 mM MgCl₂, 0.5mg/ml BSA, 200 μM each of dATP, dGTP, and dCTP, 600 μM of dUTP, 0.5 μMprimers, 1 μl of diluted Klentaq polymerase (1 μl of cold sensitiveKlentaq polymerase incubated with 10 μl of enzyme diluent), 1 unit ofuracil N-glycosylase (Amperase) and 20 μM dye D6. Polymerase chainreaction was performed on a LightCycler® (Roche Applied Systems,Indianapolis, Ind.) with an initial denaturation for 10 min at 95° C.(to denature the uracil glycosylase) followed by 45 cycles of 95° C. for3 s, cooling at 20° C./s to 58° C. (exon 9 and 11), 62° C. (exon 13), or55° C. (exon 17) for 10 s, followed by a heating 1° C./s to 75° C. for 0s. Amplicon sizes were 235 bp (exon 9), 219 bp (exon 11), 227 bp (exon13), 170 bp (exon 17). After PCR, the samples were momentarily heated to95° C. and then cooled to 40° C. on the LightCycler. The samples werethen transferred to the high resolution DNA melting analysis instrumentHR/1 (Idaho Technology, Salt Lake City, Utah). Melting analysis wasperformed as described in Example 3. All samples were run in duplicate.

Illustrative results are shown in FIGS. 29-32 in which a heterozygousSNP (FIG. 29), a homozygous deletion of 12 bp juxtaposed to an SNP (FIG.30), a heterozygous tandem duplication of 36 bp (FIG. 31), and aheterozygous deletion of 54 bp (FIG. 32) were detected byhigh-resolution amplicon melting analysis. Table 8, summarizes these andother mutations that were identified through high-resolution ampliconmelting analysis and verified by DNA sequencing.

TABLE 8 Exon number with variance detected by Base sequence changesSample high resolution established by DNA Amino acid number meltingsequencing (het/hom) alteration 1 Exon 11 missense SNP (het) V559D 2Exon 11 *54 bp deletion (het) DEL 557-574 5 Exon 11 6 bp deletion (het)DEL 558-559 6 Exon 11 *12 bp deletion + SNP (hom) DEL 554-557; K558G 7Exon 11 *36 bp duplication (het) DUP 574-585 8 Exon 11 *30 bp deletion +SNP (het) DEL 548-557; K558Q 9 Exon 11 *33 bp deletion + SNP (het) DEL547-557; K558Q 13 Exon 11 *9 bp insertion (het) INS 579-581 19 Exon 11*6 bp deletion + SNP (het) DEL 557-558; V559F 20 Exon 13 missense SNP(het) K642E 26 Exon 9 6 bp duplication (het) DUP 502-503 25 Exon 11 *27bp deletion + SNP (het) DEL 565-573; T574P 29 Exon 11 missense SNP (het)W557R Normal None None n/a control *newly identified mutations het:heterozygote hom: homozygote

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims.

All references cited herein are incorporated by reference as if fullyset forth.

1. A PCR reaction mixture for analyzing a target nucleic acid,comprising: a pair of oligonucleotide primers configured for amplifyingat least a portion of the target nucleic acid to generate an amplicon,the target nucleic acid comprising at least one of a first allele, asecond allele, a third allele, and a fourth allele; a thermostablepolymerase; an unlabeled probe comprising a nucleic acid having anucleic acid sequence completely complementary to at least a portion ofthe first allele, the unlabeled probe being blocked at its 3′-end toprevent extension of the unlabeled probe during amplification, theunlabeled probe being operable to hybridize to the first allele so as toform a first duplex and to hybridize with at least one mismatch to thesecond allele so as to form a second duplex, with at least one mismatchto the third allele so as to form a third duplex such, and with at leastone mismatch to the fourth allele so as to form a fourth duplex suchthat the unlabeled probe binds differentially to the first allele, thesecond allele, the third allele, and the fourth allele; and a dsDNAbinding dye having a percent saturation of at least 90%, the dsDNAbinding dye operable to bind to any of the first duplex, second duplex,third duplex, or fourth duplex that are present in the solution, thedsDNA binding dye being provided at a concentration suitable fordistinguishing between the first duplex, second duplex, third duplex,and fourth duplex by melting curve analysis of a signal produced by thedsDNA binding dye during melting of first duplex, second duplex, thirdduplex, or fourth duplex without the dsDNA binding dye redistributingenough during melting to obscure lower temperature melting transitions,wherein at the concentration provided, the dsDNA binding dye does notsignificantly inhibit amplification of the target nucleic acid, whereinthe unlabeled probe and the dsDNA binding dye are free in solution, theunlabeled probe and dsDNA binding dye being operable for detecting adouble-stranded nucleic acid using the dsDNA binding dye withoutimmobilizing the unlabeled probe or the amplicon, the double-strandednucleic acid comprising a strand of the amplicon hybridized to theunlabeled probe.
 2. The PCR reaction mixture of claim 1, wherein thetarget nucleic acid comprises only the first allele and melting of thefirst duplex provides a melting transition indicative of the only thefirst duplex.
 3. The PCR reaction mixture of claim 1, wherein the firstallele, the second allele, the third allele, and the fourth allelediffer from each other at a single nucleotide.
 4. The PCR reactionmixture of claim 1, wherein the first allele comprises an insertion. 5.The PCR reaction mixture of claim 1, wherein the second allele comprisesa deletion.
 6. The PCR reaction mixture of claim 1, wherein the firstprimer is provided in a molar amount greater than the second primer. 7.A method of quantifying an amount of a target nucleic acid, comprising:(a) providing a PCR reaction mixture comprising a pair ofoligonucleotide primers configured for amplifying at least a portion ofthe target nucleic acid to generate an amplicon; a thermostablepolymerase; a fluorescent dye; and a probe comprising a nucleic acidhaving a nucleic acid sequence complementary to at least a portion ofthe amplicon such that hybridizing forms a probe:target duplex, theprobe being blocked at its 3′-end to prevent extension of the unlabeledprobe during amplification, (b) thermocycling the reaction mixturethrough a specified number of cycles; (c) monitoring fluorescence duringa melting transition of the probe:target duplex to generate afluorescence curve; and (d) quantifying the amount of the target nucleicacid using the fluorescence curve.
 8. The method of claim 7, wherein theprobe is an unlabeled probe and the fluorescent dye is a saturation dye.9. The method of claim 8, wherein the fluorescent dye has a percentsaturation of at least 90%.
 10. The method of claim 7, wherein step (d)is performed by using a difference between fluorescence at a Tm of theprobe:target duplex and fluorescence at a temperature just below themelting transition of the probe:target duplex.
 11. The method of claim7, wherein step (c) includes generating a curve during the meltingtransition of the probe:target duplex and step (d) is performed using anarea under the curve.